Benzoxazines, benzothiazines, and related compounds having NOS inhibitory activity

ABSTRACT

The present invention features benzoxazines, benzothiazines, and related compounds that inhibit nitric oxide synthase (NOS), particularly those that selectively inhibit neuronal nitric oxide synthase (nNOS) in preference to other NOS isoforms. Exemplary compounds are of the formula: 
                         
The NOS inhibitors of the invention, alone or in combination with other pharmaceutically active agents, can be used for treating or preventing various medical conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/498,185, filed Jul. 6, 2009, now U.S. Pat. No. 8,106,043, whichclaims benefit of U.S. Provisional Application No. 61/133,887, filedJul. 3, 2008, both of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to the fields of benzoxazines,benzothiazines, and related compounds and to their medical use.

Nitric oxide (NO) has diverse roles both in normal and pathologicalprocesses, including the regulation of blood pressure, inneurotransmission, and in the macrophage defense systems (Snyder, S. H.and Bredt, D. S., Scientific American, May; 266(5) 1992: 68). NO issynthesized by three isoforms of nitric oxide synthase, a constitutiveform in endothelial cells (eNOS), a constitutive form in neuronal cells(nNOS), and an inducible form found in macrophage cells (iNOS). Theseenzymes are homodimeric proteins that catalyze a five-electron oxidationof L-arginine, yielding NO and citrulline. The role of NO produced byeach of the NOS isoforms is quite unique. Overstimulation oroverproduction of individual NOS isoforms, especially nNOS and iNOS,plays a role in several disorders, including septic shock, arthritis(Boughton-Smith et al., IDrugs 1:321-334, 1998 and Cochrane et al., Med.Res. Rev. 16: 547-563, 1996), diabetes, ischemia-reperfusion injury,pain (Larson et al., Pain 86:103-111, 2000), and variousneurodegenerative diseases (Kerwin et al., J. Med. Chem. 38:4343, 1995),while eNOS inhibition leads to unwanted effects such as enhanced whitecell and platelet activation, hypertension, and increased atherogenesis(Valance et al., Nature Rev. Drug Disc. 1:939, 2002).

NOS inhibitors have the potential to be used as therapeutic agents inmany disorders. However, the preservation of physiologically importantnitric oxide synthase function suggests the desirability of thedevelopment of isoform-selective inhibitors that preferentially inhibitnNOS, or nNOS and iNOS, over eNOS. Specifically, selective NOSinhibitors, particularly for nNOS or iNOS, are candidates for use in thetreatment of chronic pain states such as neuropathic pain, chronictension type headache or transformed migraine wherein the pain stateresults from the persistence of peripheral and/or central sensitization.

SUMMARY OF THE INVENTION

The invention features a compound having the formula:

wherein,

Q is —O—(CHR⁶)₁₋₃ or —S—(CHR⁶)₁₋₃—;

R¹ and each R⁶ is, independently, H, optionally substituted C₁₋₆ alkyl,optionally substituted C₁₋₄ alkaryl, optionally substituted C₁₋₄alkheterocyclyl, optionally substituted C₂₋₉ heterocyclyl, optionallysubstituted C₃₋₈ cycloalkyl, optionally substituted C₁₋₄ alkcycloalkylor —(CR^(1A)R^(1B))_(n)NR^(1C)R^(1D);

R^(1A) and R^(1B) are, independently, H, hydroxy, halo (e.g., fluoro),optionally substituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy,optionally substituted C₁₋₄ alkcycloalkyl, optionally substituted C₁₋₄alkaryl, optionally substituted C₁₋₄ alkheterocyclyl, optionallysubstituted C₁₋₄ alkheteroaryl, optionally substituted C₃₋₈ cycloalkyl,or optionally substituted C₂₋₉ heterocyclyl, or R^(1A) and R^(1B)combine to form ═O;

R^(1C) and R^(1D) are, independently, H, hydroxy, optionally substitutedC₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, optionally substitutedC₁₋₄ alkcycloalkyl, optionally substituted C₁₋₄ alkaryl, optionallysubstituted C₁₋₄ alkheterocyclyl, optionally substituted C₁₋₄alkheteroaryl, optionally substituted C₃₋₈ cycloalkyl, optionallysubstituted C₂₋₉ heterocyclyl, or an N-protecting group, or R^(1C) andR^(1D) combine to form an optionally substituted C₂₋₉ heterocyclyl or anN-protecting group;

n is an integer between 1-6;

each of R² and R³ is, independently, H, hal, optionally substituted C₁₋₆alkyl, optionally substituted C₆₋₁₀ aryl, optionally substituted C₁₋₆alkaryl, optionally substituted C₂₋₉ heterocyclyl, hydroxy, optionallysubstituted C₁₋₆ alkoxy, optionally substituted C₁₋₆ thioalkoxy,(CH₂)_(r2)NHC(NH)R^(2A), or (CH₂)_(r2)NHC(S)NHR^(2A), or optionallysubstituted C₁₋₄ alkheterocyclyl,

wherein r2 is an integer from 0 to 2, R^(2A) is optionally substitutedC₁₋₆ alkyl, optionally substituted C₆₋₁₀ aryl, optionally substitutedC₁₋₄ alkaryl, optionally substituted C₂₋₉ heterocyclyl, optionallysubstituted C₁₋₄ alkheterocyclyl, optionally substituted C₁₋₆thioalkoxy, optionally substituted C₁₋₄ thioalkaryl, optionallysubstituted aryloyl, optionally substituted C₁₋₄ thioalkheterocyclyl, oroptionally substituted amino;

each of R⁴ and R⁵ is independently H, hal, (CH₂)_(r2)NHC(NH)R^(2A), or(CH₂)_(r2)NHC(S)NHR^(2A);

wherein Y¹ and Y² are each H, or Y¹ and Y² together are ═O, or Y¹ and Y²are independently H, optionally substituted C₁₋₆ alkyl, optionallysubstituted C₆₋₁₀ aryl, optionally substituted C₁₋₆ alkaryl, optionallysubstituted C₂₋₉ heterocyclyl, hydroxy, optionally substituted C₁₋₆alkoxy, optionally substituted C₁₋₆ thioalkoxy, or optionallysubstituted C₁₋₄ alkheterocyclyl;

wherein one and only one of R², R³, R⁴, and R⁵ is(CH₂)_(r2)NHC(NH)R^(2A) or (CH₂)_(r2)NHC(S)NHR^(2A);

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, one of R¹ and R⁶ is not H.

In some embodiments, R⁶ is H.

In some embodiments, R^(1C) and R^(1D) are, independently, H, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, optionallysubstituted C₁₋₄ alkcycloalkyl, optionally substituted C₁₋₄ alkaryl,optionally substituted C₁₋₄ alkheterocyclyl, optionally substituted C₁₋₄alkheteroaryl, optionally substituted C₃₋₈ cycloalkyl, optionallysubstituted C₂₋₉ heterocyclyl, or an N-protecting group, or R^(1C) andR^(1D) combine to form an optionally substituted C₂₋₉ heterocyclyl or anN-protecting group

In some embodiments, Y¹ and Y² are each H, or Y¹ and Y² together are ═O,or Y¹ and Y² are independently H, optionally substituted C₁₋₆ alkyl,optionally substituted C₆₋₁₀ aryl, optionally substituted C₁₋₆ alkaryl,optionally substituted C₂₋₉ heterocyclyl, optionally substituted C₁₋₆alkoxy, optionally substituted C₁₋₆ thioalkoxy, or optionallysubstituted C₁₋₄ alkheterocyclyl.

In some embodiments, R², R³, R⁴, or R⁵ may have the formula:

In further embodiments, R^(2A) has the formula:

wherein

each of X¹, X², X⁴, and X⁵ is independently selected from O, S, NR⁷, N,or CR⁸; X³ is selected from N or C;

R⁷ is H, optionally substituted C₁₋₆ alkyl, or an N-protecting group;

R⁸ is H, hal, optionally substituted C₁₋₆ alkyl, hydroxy, optionallysubstituted C₁₋₆ alkoxy, or optionally substituted C₁₋₆ thioalkoxy,

wherein at least one of X¹, X², X⁴, and X⁵ is not CR⁸. In particular,R^(2A) may have the formula:

whereineach of X¹ and X² is independently selected from O, S, NH, N, or CH; andwherein at least one of X¹ and X² is not CH. In certain otherembodiments, X¹ is CH, and X² is S. In still other embodiments, X¹ isCH, and X² is O.

In some embodiments, the compound has a structure selected from:

wherein

one of R⁴ and R⁵ has the following structure:

and wherein X² is O or S.

In some embodiments, Q is O—(CHR⁶)₁₋₂ or S—(CHR⁶)₁₋₂; and R¹ and each R⁶are, independently, H, optionally substituted C₁₋₆ alkyl, optionallysubstituted C₁₋₄ alkaryl, optionally substituted C₁₋₄ alkheterocyclyl,or optionally substituted C₂₋₉ heterocyclyl.

In other embodiments, Y¹ and Y² are each H, and Q is O—CHR⁶, O—(CHR⁶)₂,or O—(CHR⁶)₃; or Y¹ and Y² together are ═O, and Q is O—CHR⁶, O—(CHR⁶)₂,or O—(CHR⁶)₃.

In other embodiments, Y¹ and Y² are each H, and Q is S—CHR⁶, S—(CHR⁶)₂,or S—(CHR⁶)₃; or Y¹ and Y² together are ═O, and Q is S—CHR⁶, S—(CHR⁶)₂,or S—(CHR⁶)₃.

In some embodiments, R¹ is optionally substituted C₁₋₆ alkyl, optionallysubstituted C₂₋₉ heterocyclyl, optionally substituted C₁₋₄alkheterocyclyl, optionally substituted C₃-C₈ cycloalkyl, or—(CR^(1A)R^(1B))_(n)NR^(1C)R^(1D). In other embodiments, R¹ isaminoC₁₋₆alkyl. In still other embodiments, R¹ is optionally substitutedC₁₋₄ alkheterocyclyl, where the heterocyclyl is a 5- or 6-memberedcyclic amine. In specific embodiments, a 5-membered cyclic amine issubstituted with a carboxylic acid, ester (e.g., a C₁₋₆ ester), oramide. In some embodiments, R¹ is optionally substituted C₂₋₉heterocyclyl. In other embodiments, the heterocyclyl is optionallysubstituted pyrrolidinyl or optionally substituted piperidinyl, e.g.,

where R⁹ is H, optionally substituted C₁₋₆ alkyl or an N-protectinggroup. In specific embodiments R⁹ is H. In other embodiments, R¹ is—(CR^(1A)R^(1B))_(n)NR^(1C)R^(1D). In certain embodiments, R^(1A) andR^(1B) are each H. In further embodiments, n is 2 or 3. In otherembodiments, NR^(1C) is H, and NR^(1D) is —CH₃, —CH₂CH₃, —(CH₂)₂OH, or—CH₂CO₂H. In still other embodiments, R¹ is —CH₂CH₂N(CH₃)₂. In otherembodiments, R¹ is —CH₂CH₂NHCH₃.

In certain embodiments, R¹ is an optionally substituted C₃-C₈cycloalkyl. In certain embodiments, the C₃-C₈ cycloalkyl is substitutedby an optionally substituted amino

In other embodiments, one of R⁴ or R⁵ is H or F.

Specific preferred compounds of formula (I) include:

or a pharmaceutically acceptable salt thereof, e.g., the dihydrochloridesalt.

The invention also features a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt orprodrug thereof, and a pharmaceutically acceptable excipient.

Preferably, a compound of the invention selectively inhibits neuronalnitric oxide synthase (nNOS), particularly over endothelial nitric oxidesynthase (eNOS) or inducible nitric oxide synthase (iNOS) or both.

Preferably, the IC₅₀ or K_(i) value observed for the compound is atleast 2 times lower for nNOS than for eNOS and/or iNOS. More preferably,the IC₅₀ or K_(i) value is at least 5, 20, 50, or 100 times lower (i.e.,more potent in nNOS). In one embodiment, the IC₅₀ or K_(i) value isbetween 2 times and 100 times lower. In another embodiment, the IC₅₀ orK_(i) in eNOS is greater than 10 μM. More preferably eNOS IC₅₀ isgreater than 20 μM, most preferably eNOS IC₅₀ or Ki is greater than 30μM, as a threshold level of eNOS may be needed to avoid any direct eNOSmediated constriction of human vascular tissue.

In another embodiment of the invention, the IC₅₀ or K_(i) of iNOS andnNOS is between 2 and greater than 100 times lower than eNOS. Mostpreferably, the IC₅₀ or K_(i) of nNOS or iNOS is at least 20, 50, or 100fold lower. In another embodiment, compounds of the invention areselective nNOS inhibitors.

The invention further features a method of treating or preventing acondition in a mammal, such as a human, caused by the action of nitricoxide synthase (NOS), e.g., nNOS, that includes administering aneffective amount of a compound of the invention to the mammal. Examplesof such conditions include: headache (e.g., migraine headache (with orwithout aura), chronic tension type headache (CTTH), migraine withallodynia, medication overuse headache, cluster headache, chronicheadache, or transformed migraine); neuropathic pain (AIDS associatedpainful neuropathy, central post-stroke pain (CPSP), diabeticneuropathy, chemotherapy induced neuropathic pain (e.g., paclitaxel,cis-Platin, Doxorubicin etc.), postherpetic neuralgia, or trigeminalneuralgia), chronic inflammatory pain (e.g., pain that results fromosteoarthritis, rheumatoid arthritis, ankylosing spondylitis, psoriaticarthritis, undifferentiated spondyloarthropathy, or reactive arthritis);visceral pain; neuroinflammation; medication-induced hyperalgesia and/orallodynia (e.g., opioid-induced hyperalgesia/allodynia or triptan(5-HT_(1D/1B) agonists)-induced hyperalgesia or allodynia); acute pain(optionally in combination with an opioid treatment); chronic pain; bonecancer pain; chemical dependencies or addictions (e.g., drug addiction;cocaine addiction; nicotine addiction; metamphetamine-inducedneurotoxicity; ethanol tolerance, dependence, or withdrawal; ormorphine/opioid induced tolerance, dependence, hyperalgesia, orwithdrawal); CNS disorders (e.g., epilepsy, anxiety, depression (aloneor in combination), attention deficit hyperactivity disorder (ADHD),psychosis, or dementia); neurodegenerative diseases or nerve injury(e.g., acute spinal cord injury, AIDS associated dementia, Parkinson'sdisease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS),Huntington's disease, multiple sclerosis, neurotoxicity, or headtrauma); cardiovascular related conditions (e.g., stroke, coronaryartery bypass graft (CABG) associated neurological damage, hypothermiccardiac arrest (HCA), post-stroke pain, cardiogenic shock, reperfusioninjury, or vascular dementia); or gastrointestinal disorders (e.g.,ileostomy-associated diarrhea or dumping syndrome).

In other embodiments, a compound of the invention can be used for thetreatment of chronic pain with central sensitization. In otherembodiments, the pain with components of central sensitization isneuropathic pain. In other embodiments, the neuropathic pain is selectedfrom postherpetic neuralgia, diabetic neuropathy, central (thalamic)pain, post-stroke pain, HIV-associated pain, phantom limb pain,neuropathic pain resulting from post-surgical trauma or nerve injury,and chemotherapy-induced neuropathies.

In other embodiments, a compound of the invention can be used for thetreatment of chronic inflammatory pain. In other embodiments, thechronic inflammatory pain relates to ankylosing spondylitis, reactivearthritis (Reiter's syndrome), psoriatic arthritis, undifferentiatedspondyloarthropathy, rheumatoid arthritis, and osteoarthritis.

In other embodiments, a compound of the invention can be used for thetreatment of headaches with underlying mechanisms of centralsensitization. In other embodiments, headache is selected from migraine,chronic tension type headache (CTTH), cluster headache, transformedmigraine, and medication overuse headache.

Still other embodiments include use of a compound of the invention fortreatment of interstitial cystitis or opiate withdrawal or migraineprophylaxis.

A compound of the invention can also be used in combination with one ormore other therapeutic agents for the prevention or treatment of one ofthe aforementioned conditions.

Exemplary agents useful in combination with a compound of the invention,include opioids, antidepressants, antiepileptics, non-steroidalanti-inflammatory drugs (NSAIDs), antiarrhythmics, GABA-B antagonists,alpha-2-adrenergic receptor agonists, serotonin 5HT_(1B/1D) agonists,N-methyl-D-aspartate antagonists, cholecystokinin B antagonists,substance P antagonists (NK1), anti-inflammatory compounds,DHP-sensitive L-type calcium channel antagonists,omega-conotoxin-sensitive N-type calcium channel antagonists, P/Q-typecalcium channel antagonists, adenosine kinase antagonists, adenosinereceptor A₁ agonists, adenosine receptor A_(2a) antagonists, adenosinereceptor A₃ agonists, adenosine deaminase inhibitors, adenosinenucleoside transport inhibitors, vanilloid VR1 receptor agonists,cannabinoid CB1/CB2 agonists, AMPA receptor antagonists, kainatereceptor antagonists, sodium channel blockers (e.g., Nav1.8 blocker forneuropathic pain), nicotinic acetylcholine receptor agonists, a K_(ATP)potassium channel, K_(v1.4) potassium channel, Ca²⁺-activated potassiumchannel, SK potassium channel, BK potassium channel, IK potassiumchannel, or KCNQ2/3 potassium channel opening agents, muscarinic M3antagonists, muscarinic M1 agonists, muscarinic M2/M3 partialagonists/antagonists, and antioxidants. Specific examples of therapeuticagents that are useful in combination with a compound of the inventionare listed in Table 1. Other classes include CB1/CB2 agonists, e.g.,dexanabinol (HU-211), fatty acid amide hydrolase inhibitors, P2Xpurinergic blockers, and NGF antagonists.

TABLE 1 Therapeutic agents useful in combination with compounds of theinvention Class Examples Opioid alfentanil, butorphanol, buprenorphine,codeine, dextromoramide, dextropropoxyphene, dezocine, dihydrocodeine,diphenoxylate, etorphine, fentanyl, hydrocodone, hydromorphone,ketobemidone, levorphanol, levomethadone, methadone, meptazinol,morphine, morphine-6-glucuronide, nalbuphine, naloxone, oxycodone,oxymorphone, pentazocine, pethidine, piritramide, remifentanil,sulfentanyl, tilidine, or tramadol Antidepressant alaproclate,citalopram, chlomipramine, escitalopram, femoxetine, (selectivefluoxetine, fluvoxamine, paroxetine, sertraline, or zimelidine serotoninre- uptake inhibitor) Antidepressant adinazolam, amiltriptylinoxide,amineptine, amoxapine, atomoxetine, (norepinephrine- bupropion,butriptyline, desipramine, doxepin, desipramine, reuptake maprotiline,nortriptyline (desmethylamitriptyline), demexiptiline, inhibitor)dothiepin, fluacizine, imipramine, imipramine oxide, iprindole,lofepramine, maprotiline, melitracen, metapramine, norclolipramine,noxiptilin, opipramol, perlapine, pizotyline, propizepine, quinupramine,reboxetine, or tianeptine, tomoxetine, trimipramine or viloxazineAntidepressant duloxetine, milnacipran, mirtazapine, nefazodone,venlafaxine, or (dual serotonin/ desvenlafaxine norepinephrine reuptakeinhibitor) Antidepressant amiflamine, iproniazid, isocarboxazid, M-3-PPC(Draxis), (monoamine moclobemide, pargyline, phenelzine,tranylcypromine, or vanoxerine oxidase inhibitor) Antidepressantbazinaprine, befloxatone, brofaromine, cimoxatone, or clorgyline(reversible monoamine oxidase type A inhibitor) Antidepressantamitriptyline, amoxapine, buriptyline, clomipramine, desipramine,(tricyclic) debenzepin, dothiepin, doxepin, imipramine, iprindole, ,lofepramine, melitracen, opipramol, nortryptyline, protriptyline, ortrimipramine Antidepressant adinazolam, alaproclate, amineptine,amitriptyline/chlordiazepoxide (other) combination, atipamezole,azamianserin, bazinaprine, befuraline, bifemelane, binodaline,bipenamol, brofaromine, caroxazone, cericlamine, cianopramine,cimoxatone, citalopram, clemeprol, clovoxamine, dazepinil, deanol,demexiptiline, dibenzepin, dothiepin, droxidopa, enefexine, estazolam,etoperidone, femoxetine, fengabine, fezolamine, fluotracen, idazoxan,indalpine, indeloxazine, iprindole, levoprotiline, lithium, litoxetine;lofepramine, medifoxamine, metapramine, metralindole, mianserin,milnacipran, minaprine, mirtazapine, montirelin, nebracetam, nefopam,nialamide, nomifensine, norfluoxetine, orotirelin, oxaflozane,pinazepam, pirlindone, pizotyline, ritanserin, rolipram, sercloremine,setiptiline, sibutramine, sulbutiamine, sulpiride, teniloxazine,thozalinone, thymoliberin, tianeptine, tiflucarbine, trazodone,tofenacin, tofisopam, toloxatone, tomoxetine, veralipride, viloxazine,viqualine, zimelidine, or zometapine Antiepileptic carbamazepine,flupirtine, gabapentin, lamotrigine, oxcarbazepine, phenytoin,pregabalin, retigabine, topiramate, or valproate Non-steroidalacemetacin, aspirin, celecoxib, deracoxib, diclofenac, diflunisal, anti-ethenzamide, etofenamate, etoricoxib, fenoprofen, flufenamic acid,inflammatory flurbiprofen, lonazolac, lornoxicam, ibuprofen,indomethacin, drug (NSAID) isoxicam, kebuzone, ketoprofen, ketorolac,naproxen, nabumetone, niflumic acid, sulindac, tolmetin, piroxicam,meclofenamic acid, mefenamic acid, meloxicam, metamizol, mofebutazone,oxyphenbutazone, parecoxib, phenidine, phenylbutazone, piroxicam,propacetamol, propyphenazone, rofecoxib, salicylamide, suprofen,tiaprofenic acid, tenoxicam, valdecoxib, 4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide, 2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone, or 2-(3,5-difluorophenyl)-3-[4-(methylsulfonyl)phenyl]-2-cyclopenten-1-one).5HT_(1B/1D) agonist eletriptan, frovatriptan, naratriptan, rizatriptan,sumatriptan, almotriptan, donitriptan, or zolmitriptan Anti- aspirin,celecoxib, cortisone, deracoxib, diflunisal, etoricoxib, inflammatoryfenoprofen, ibuprofen, ketoprofen, naproxen, prednisolone, sulindac,compounds tolmetin, piroxicam, mefenamic acid, meloxicam,phenylbutazone, rofecoxib, suprofen, valdecoxib,4-(4-cyclohexyl-2-methyloxazol-5- yl)-2-fluorobenzenesulfonamide,N-[2-(cyclohexyloxy)-4- nitrophenyl]methanesulfonamide,2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone, or 2-(3,5-difluorophenyl)-3-[4-(methylsulfonyl)phenyl]-2-cyclopenten-1-one N-methyl-D- amantadine;aptiganel; besonprodil; budipine; conantokin G; aspartate delucemine;dexanabinol; dextromethorphan; antagonist and dextropropoxyphen;felbamate; fluorofelbamate; gacyclidine; glycine; other glutamateipenoxazone; kaitocephalin; ketamine; ketobemidone; lanicemine; receptorlicostinel; midafotel; memantine; D-methadone; D-morphine; antagonists(e.g., milnacipran; neramexane; orphenadrine; remacemide; sulfazocine;AMPA/kainite FPL-12,495 (racemide metabolite); topiramate;(αR)-α-amino-5- (GluR5),chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoic acid; 1- MGluR,and aminocyclopentane-carboxylic acid; [5-(aminomethyl)-2-[[[(5S)-9-iGluR) chloro-2,3,6,7-tetrahydro-2,3-dioxo-1H-,5H-pyrido[1,2,3-(Medicinal de]quinoxalin-5-yl]acetyl]amino]phenoxy]-acetic acid;α-amino-2- Research (2-phosphonoethyl)-cyclohexanepropanoic acid;α-amino-4- Reviews, 2007; (phosphonomethyl)-benzeneacetic acid;(3E)-2-amino-4- 27(2): 239-278 (phosphonomethyl)-3-heptenoic acid;3-[(1E)-2-carboxy-2- and Basic &phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylic acid; 8-chloro-Clinical. 2,3-dihydropyridazino[4,5-b]quinoline-1,4-dione 5-oxide saltwith 2- Parmacol. hydroxy-N,N,N-trimethyl-ethanaminium; N′-[2-chloro-5-Toxicol. 2005,(methylthio)phenyl]-N-methyl-N-[3-(methylthio)phenyl]-guanidine; 97:202-213) N′-[2-chloro-5-(methylthio)phenyl]-N-methyl-N-[3-[(R)-methylsulfinyl]phenyl]-guanidine; 6-chloro-2,3,4,9-tetrahydro-9-methyl-2,3-dioxo-1H-indeno[1,2-b]pyrazine-9-acetic acid; 7-chlorothiokynurenic acid; (3S, 4aR, 6S, 8aR)-decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylic acid; (−)-6,7-dichloro-1,4-dihydro-5-[3-(methoxymethyl)-5-(3-pyridinyl)-4-H-1,2,4-triazol-4-yl]-2,3-quinoxalinedione; 4,6-dichloro-3-[(E)-(2-oxo-1-phenyl-3-pyrrolidinylidene)methyl]-1H-indole-2-carboxylic acid; (2R,4S)-rel-5,7-dichloro-1,2,3,4-tetrahydro-4-[[(phenylamino)carbonyl]amino]-2-quinolinecarboxylic acid; (3R,4S)-rel-3,4-dihydro-3-[4-hydroxy-4-(phenylmethyl)-1-piperidinyl-]-2H-1-benzopyran-4,7-diol; 2-[(2,3-dihydro-1H-inden-2-yl)amino]-acetamide; 1,4-dihydro-6-methyl-5-[(methylamino)methyl]-7-nitro-2,3-quinoxalinedione; [2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl]-phosphonic acid;(2R,6S)-1,2,3,4,5,6-hexahydro-3-[(2S)-2-methoxypropyl]-6,11,11-trimethyl-2,6-methano-3-benzazocin-9-ol; 2-hydroxy-5-[[(pentafluorophenyl)methyl]amino]-benzoic acid; 1-[2-(4-hydroxyphenoxy)ethyl]-4-[(4-methylphenyl)methyl]-4-piperidinol; 1-[4-(1H-imidazol-4-yl)-3-butynyl]-4-(phenylmethyl)-piperidine; 2-methyl-6-(phenylethynyl)-pyridine; 3-(phosphonomethyl)-L- phenylalanine;efenprodil, CP101606, Ro256981, or 3,6,7-tetrahydro-2,3-dioxo-N-phenyl-1H,5H-pyrido[1,2,3-de]quinoxaline-5-acetamideOther compounds useful for treating pain are described in WO/2003/034900and in U.S. Patent Publication No. 20030082225, each of which is herebyincorporated by reference.

NMDA antagonists in combination with nNOS inhibitors may be particularlyuseful in treating conditions such as inflammatory and neuropathic pain,traumatic brain injury, and Parkinson's Disease (see Drug DiscoveryToday 7(7):403-406, 2002).

Compounds of the invention may also be employed in combination with FAAHinhibitors, such as those described in: J. Med. Chem. 49:4650-4656,2006; Neuropharmacology 50:814-823, 2006; Current Opinion in ChemicalBiology 7:469-475, 2003; and Pain 109:319-327, 2004, each of which isincorporated by reference.

Asymmetric or chiral centers may exist in any of the compounds of thepresent invention. The present invention contemplates the variousstereoisomers and mixtures thereof. Individual stereoisomers ofcompounds of the present invention are prepared synthetically fromcommercially available starting materials that contain asymmetric orchiral centers or by preparation of mixtures of enantiomeric compoundsfollowed by resolution well-known to those of ordinary skill in the art.These methods of resolution are exemplified by (1) attachment of aracemic mixture of enantiomers, designated (+/−), to a chiral auxiliary,separation of the resulting diastereomers by recrystallization orchromatography and liberation of the optically pure product from theauxiliary or (2) direct separation of the mixture of optical enantiomerson chiral chromatographic columns. Alternatively, chiral compounds canbe prepared by an asymmetric synthesis that favors the preparation ofone enantiomer over the other. Alternatively a chiral pool synthesis(starting with an enantiomerically pure building block) can be usedwherein the chiral group or center is retained in the intermediate orfinal product. Enantiomers are designated herein by the symbols “R,” or“S,” depending on the configuration of substituents around the chiralatom. Alternatively, enantiomers are designated as (+) or (−) dependingon whether a solution of the enantiomer rotates the plane of polarizedlight clockwise or counterclockwise, respectively. In other cases,diastereomeric isomers such as cis and trans isomers may be separated bycolumn chromatography, chiral chromatography, or recrystallization. Insome cases, derivatization can improve the separation of these mixtures.

Geometric isomers may also exist in the compounds of the presentinvention. The present invention contemplates the various geometricisomers and mixtures thereof resulting from the arrangement ofsubstituents around a carbon-carbon double bond and designates suchisomers as of the Z or E configuration. It is also recognized that forstructures in which tautomeric forms are possible, the description ofone tautomeric form is equivalent to the description of both, unlessotherwise specified. For example, amidine structures of the formula—C(═NR^(Q))NHR^(T) and —C(NHR^(Q))═NR^(T), where R^(T) and R^(Q) aredifferent, are equivalent tautomeric structures and the description ofone inherently includes the other.

It is understood that substituents and substitution patterns on thecompounds of the invention can be selected by one of ordinary skill inthe art to provide compounds that are chemically stable and that can bereadily synthesized by techniques known in the art, as well as thosemethods set forth below, from readily available starting materials. If asubstituent is itself substituted with more than one group, it isunderstood that these multiple groups may be on the same carbon or ondifferent carbons, so long as a stable structure results.

Other features and advantages will be apparent from the followingdescription and the claims.

DEFINITIONS

The term “acyl,” or “alkanoyl,” as used interchangeably herein,represent an alkyl group, as defined herein, or hydrogen attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl, acetyl, propionyl, butanoyl and the like.Exemplary unsubstituted acyl groups include from 2 to 7 carbons.

The term “C_(x-y) alkaryl,” as used herein, represents a chemicalsubstituent of formula —RR′, where R is an alkylene group of x to ycarbons and R′ is an aryl group as defined herein. Similarly, by theterm “C_(x-y) alkheteroaryl” is meant a chemical substituent of formula—RR″, where R is an alkylene group of x to y carbons and R″ is aheteroaryl group as defined herein. Other groups preceded by the prefix“alk-” are defined in the same manner. Exemplary unsubstituted alkarylgroups are of from 7 to 16 carbons.

The term “alkcycloalkyl” represents a cycloalkyl group attached to theparent molecular group through an alkylene group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 6carbons containing one or more carbon-carbon double bonds and isexemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl,1-butenyl, 2-butenyl, and the like.

The term “alkheterocyclyl” represents a heterocyclic group attached tothe parent molecular group through an alkylene group. Exemplaryunsubstituted alkheterocyclyl groups are of from 2 to 14 carbons.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is an alkyl group of 1 to 6 carbons, unless otherwise specified.

The term “alkoxyalkyl” represents an alkyl group which is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 12 carbons.

The terms “alkyl” and the prefix “alk-,” as used herein, are inclusiveof both straight chain and branched chain saturated groups of from 1 to6 carbons, unless otherwise specified. Alkyl groups are exemplified bymethyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl,neopentyl, and the like, and may be optionally substituted with one,two, three or, in the case of alkyl groups of two carbons or more, foursubstituents independently selected from the group consisting of: (1)alkoxy of one to six carbon atoms; (2) alkylsulfinyl of one to sixcarbon atoms; (3) alkylsulfonyl of one to six carbon atoms; (4) amino;(5) aryl; (6) arylalkoxy; (7) aryloyl; (8) azido; (9) carboxaldehyde;(10) cycloalkyl of three to eight carbon atoms; (11) hal; (12)heterocyclyl; (13) (heterocycle)oxy; (14) (heterocycle)oyl; (15)hydroxy; (16) N-protected amino; (17) nitro; (18) oxo; (19) spirocyclylof three to eight carbon atoms; (20) thioalkoxy of one to six carbonatoms; (21) thiol; (22) —CO₂R^(A), where R^(A) is selected from thegroup consisting of (a) alkyl, (b) aryl, (c) alkaryl, and (d) hydrogen,where the alkylene group is of one to six carbon atoms; (23)—C(O)NR^(B)R^(C), where each of R^(B) and R^(C) is, independently,selected from the group consisting of (a) hydrogen, (b) alkyl, (c) aryland (d) alkaryl, where the alkylene group is of one to six carbon atoms;(24) —SO₂R^(D), where R^(D) is selected from the group consisting of (a)alkyl, (b) aryl and (c) alkaryl, where the alkylene group is of one tosix carbon atoms; (25) —SO₂NR^(E)R^(F), where each of R^(E) and R^(F)is, independently, selected from the group consisting of (a) hydrogen,(b) alkyl, (c) aryl and (d) alkaryl, where the alkylene group is of oneto six carbon atoms; and (26) —NR^(G)R^(H), where each of R^(G) andR^(H) is, independently, selected from the group consisting of (a)hydrogen; (b) an N-protecting group; (c) alkyl of one to six carbonatoms; (d) alkenyl of two to six carbon atoms; (e) alkynyl of two to sixcarbon atoms; (f) aryl; (g) alkaryl, where the alkylene group is of oneto six carbon atoms; (h) cycloalkyl of three to eight carbon atoms; and(i) alkcycloalkyl, where the cycloalkyl group is of three to eightcarbon atoms, and the alkylene group is of one to ten carbon atoms,wherein in one embodiment no two groups are bound to the nitrogen atomthrough a carbonyl group or a sulfonyl group.

The term “alkylene,” as used herein, represents a saturated divalenthydrocarbon group derived from a straight or branched chain saturatedhydrocarbon by the removal of two hydrogen atoms, and is exemplified bymethylene, ethylene, isopropylene, and the like.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are of from 1 to 6 carbons.

The term “alkylsulfonyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —SO₂— group. Exemplaryunsubstituted alkylsulfonyl groups are of from 1 to 6 carbons.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are of from 2 to 12carbons.

The term “alkylsulfonylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfonyl group.Exemplary unsubstituted alkylsulfonylalkyl groups are of from 2 to 12carbons.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups of from two to six carbon atoms containing acarbon-carbon triple bond and is exemplified by ethynyl, 1-propynyl, andthe like.

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group. Theterm “amino,” as used herein, represents —NH₂, —NHR^(N1), or—N(R^(N1))₂, wherein each R^(N1) is, independently, H, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), optionally substituted C₁₋₆alkyl, optionally substituted C₁₋₆ alkoxy, optionally substituted C₁₋₄alkcycloalkyl, optionally substituted C₁₋₄ alkaryl, optionallysubstituted C₁₋₄ alkheterocyclyl, optionally substituted C₁₋₄alkheteroaryl, optionally substituted C₃₋₈cycloalkyl, optionallysubstituted C₂₋₉ heterocyclyl, or an N-protecting group, or two R^(N1)combine to form an optionally substituted C₂₋₉ heterocyclyl, or anN-protecting group, and wherein each R^(N2) is, independently, H, anoptionally substituted alkyl group, or an optionally substituted arylgroup. In a preferred embodiment, amino is —NH2, or —NHR^(N1), whereineach R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂, SO₂OR^(N2),SO₂R^(N2), SOR^(N2), an optionally substituted alkyl group, or anoptionally substituted aryl group, and each R^(N2) can be H, anoptionally substituted alkyl group, or an optionally substituted arylgroup.

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group.

The term “aryl,” as used herein, represents a mono- or bicycliccarbocyclic ring system having one or two aromatic rings and isexemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, and the like,and may be optionally substituted with one, two, three, four, or fivesubstituents independently selected from the group consisting of: (1)alkanoyl of one to six carbon atoms; (2) alkyl of one to six carbonatoms; (3) alkoxy of one to six carbon atoms; (4) alkoxyalkyl, where thealkyl and alkylene groups are independently of one to six carbon atoms;(5) alkylsulfinyl of one to six carbon atoms; (6) alkylsulfinylalkyl,where the alkyl and alkylene groups are independently of one to sixcarbon atoms; (7) alkylsulfonyl of one to six carbon atoms; (8)alkylsulfonylalkyl, where the alkyl and alkylene groups areindependently of one to six carbon atoms; (9) aryl; (10) amino; (11)aminoalkyl of one to six carbon atoms; (12) heteroaryl; (13) alkaryl,where the alkylene group is of one to six carbon atoms; (14) aryloyl;(15) azido; (16) azidoalkyl of one to six carbon atoms; (17)carboxaldehyde; (18) (carboxaldehyde)alkyl, where the alkylene group isof one to six carbon atoms; (19) cycloalkyl of three to eight carbonatoms; (20) alkcycloalkyl, where the cycloalkyl group is of three toeight carbon atoms and the alkylene group is of one to ten carbon atoms;(21) hal; (22) haloalkyl of one to six carbon atoms; (23) heterocyclyl;(24) (heterocyclyl)oxy; (25) (heterocyclyl)oyl; (26) hydroxy; (27)hydroxyalkyl of one to six carbon atoms; (28) nitro; (29) nitroalkyl ofone to six carbon atoms; (30) N-protected amino; (31) N-protectedaminoalkyl, where the alkylene group is of one to six carbon atoms; (32)oxo; (33) thioalkoxy of one to six carbon atoms; (34) thioalkoxyalkyl,where the alkyl and alkylene groups are independently of one to sixcarbon atoms; (35) —(CH₂)_(q)CO₂R^(A), where q is an integer of fromzero to four, and R^(A) is selected from the group consisting of (a)alkyl, (b) aryl, (c) alkaryl, and (d) hydrogen, where the alkylene groupis of one to six carbon atoms; (36) —(CH₂)_(q)CONR^(B)R^(C), where q isan integer of from zero to four and where R^(B) and R^(C) areindependently selected from the group consisting of (a) hydrogen, (b)alkyl, (c) aryl, and (d) alkaryl, where the alkylene group is of one tosix carbon atoms; (37) —(CH₂)_(q)SO₂R^(D), where q is an integer of fromzero to four and where R^(D) is selected from the group consisting of(a) alkyl, (b) aryl, and (c) alkaryl, where the alkylene group is of oneto six carbon atoms; (38) —(CH₂)_(q)SO₂NR^(E)R^(F), where q is aninteger of from zero to four and where each of R^(E) and R^(F) is,independently, selected from the group consisting of (a) hydrogen, (b)alkyl, (c) aryl, and (d) alkaryl, where the alkylene group is of one tosix carbon atoms; (39) —(CH₂)_(q)NR^(G)R^(H), where q is an integer offrom zero to four and where each of R^(G) and R^(H) is, independently,selected from the group consisting of (a) hydrogen; (b) an N-protectinggroup; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to sixcarbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g)alkaryl, where the alkylene group is of one to six carbon atoms; (h)cycloalkyl of three to eight carbon atoms; and (i) alkcycloalkyl, wherethe cycloalkyl group is of three to eight carbon atoms, and the alkylenegroup is of one to ten carbon atoms, wherein in one embodiment no twogroups are bound to the nitrogen atom through a carbonyl group or asulfonyl group; (40) thiol; (41) perfluoroalkyl; (42) perfluoroalkoxy;(43) aryloxy; (44) cycloalkoxy; (45) cycloalkylalkoxy; and (46)arylalkoxy.

The term “arylalkoxy,” as used herein, represents an alkaryl groupattached to the parent molecular group through an oxygen atom. Exemplaryunsubstituted arylalkoxy groups are of from 7 to 16 carbons.

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified.

The term “aryloyl,” as used herein, represent an aryl group that isattached to the parent molecular group through a carbonyl group.Exemplary unsubstituted aryloyl groups are of 7 or 11 carbons.

The term “azido” represents an N₃ group.

The term “azidoalkyl” represents an azido group attached to the parentmolecular group through an alkyl group.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents a CHO group.

The term “carboxaldehydealkyl” represents a carboxyaldehyde groupattached to the parent molecular group through an alkylene group.

The term “chronic tension type headache” (CTTH), as used herein, means atension type headache that meets the diagnostic criteria defined by theInternational Headache Society Classification, 2^(nd) Edition (ICHD-2),e.g., a headache that occurs at least 15 days per month on average for aperiod of >3 months (at least 180 days per year).

The term “chronic migraine,” which is also called “transformedmigraine,” as used herein, refers to the definition provided in theICHD-2, and is “migraine headache occurring on 15 or more days per monthfor more than 3 months in the absence of medication overuse.” Clinicalguidelines for the diagnosis of chronic migraine can be found, forexample, in the ICHD-2 and in Oleson et al., Cephalalgia, 26(6):742-746,2006.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group of from three toeight carbons, unless otherwise specified, and is exemplified bycyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,bicyclo[2.2.1.]heptyl and the like. The cycloalkyl groups of thisinvention can be optionally substituted with (1) alkanoyl of one to sixcarbon atoms; (2) alkyl of one to six carbon atoms; (3) alkoxy of one tosix carbon atoms; (4) alkoxyalkyl, where the alkyl and alkylene groupsare independently of one to six carbon atoms; (5) alkylsulfinyl of oneto six carbon atoms; (6) alkylsulfinylalkyl, where the alkyl andalkylene groups are independently of one to six carbon atoms; (7)alkylsulfonyl of one to six carbon atoms; (8) alkylsulfonylalkyl, wherethe alkyl and alkylene groups are independently of one to six carbonatoms; (9) aryl; (10) amino; (11) aminoalkyl of one to six carbon atoms;(12) heteroaryl; (13) alkaryl, where the alkylene group is of one to sixcarbon atoms; (14) aryloyl; (15) azido; (16) azidoalkyl of one to sixcarbon atoms; (17) carboxaldehyde; (18) (carboxaldehyde)alkyl, where thealkylene group is of one to six carbon atoms; (19) cycloalkyl of threeto eight carbon atoms; (20) alkcycloalkyl, where the cycloalkyl group isof three to eight carbon atoms and the alkylene group is of one to tencarbon atoms; (21) hal; (22) haloalkyl of one to six carbon atoms; (23)heterocyclyl; (24) (heterocyclyl)oxy; (25) (heterocyclyl)oyl; (26)hydroxy; (27) hydroxyalkyl of one to six carbon atoms; (28) nitro; (29)nitroalkyl of one to six carbon atoms; (30) N-protected amino; (31)N-protected aminoalkyl, where the alkylene group is of one to six carbonatoms; (32) oxo; (33) thioalkoxy of one to six carbon atoms; (34)thioalkoxyalkyl, where the alkyl and alkylene groups are independentlyof one to six carbon atoms; (35) —(CH₂)_(q)CO₂R^(A), where q is aninteger of from zero to four, and R^(A) is selected from the groupconsisting of (a) alkyl, (b) aryl, (c) alkaryl, and (d) hydrogen, wherethe alkylene group is of one to six carbon atoms; (36)—(CH₂)_(q)CONR^(B)R^(C), where q is an integer of from zero to four andwhere R^(B) and R^(C) are independently selected from the groupconsisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d) alkaryl, wherethe alkylene group is of one to six carbon atoms; (37)—(CH₂)_(q)SO₂R^(D), where q is an integer of from zero to four and whereR^(D) is selected from the group consisting of (a) alkyl, (b) aryl, and(c) alkaryl, where the alkylene group is of one to six carbon atoms;(38) —(CH₂)_(q)SO₂NR^(E)R^(F), where q is an integer of from zero tofour and where each of R^(E) and R^(F) is, independently, selected fromthe group consisting of (a) hydrogen, (b) alkyl, (c) aryl, and (d)alkaryl, where the alkylene group is of one to six carbon atoms; (39)—(CH₂)_(q)NR^(G)R^(H), where q is an integer of from zero to four andwhere each of R^(G) and R^(H) is, independently, selected from the groupconsisting of (a) hydrogen; (b) an N-protecting group; (c) alkyl of oneto six carbon atoms; (d) alkenyl of two to six carbon atoms; (e) alkynylof two to six carbon atoms; (f) aryl; (g) alkaryl, where the alkylenegroup is of one to six carbon atoms; (h) cycloalkyl of three to eightcarbon atoms; and (i) alkcycloalkyl, where the cycloalkyl group is ofthree to eight carbon atoms, and the alkylene group is of one to tencarbon atoms, with the proviso that no two groups are bound to thenitrogen atom through a carbonyl group or a sulfonyl group; (40) thiol;(41) perfluoroalkyl; (42) perfluoroalkoxy; (43) aryloxy; (44)cycloalkoxy; (45) cycloalkylalkoxy; and (46) arylalkoxy.

The terms “cycloalkyloxy” or “cycloalkoxy”, as used interchangeablyherein, represent a cycloalkyl group, as defined herein, attached to theparent molecular group through an oxygen atom. Exemplary unsubstitutedcycloalkyloxy groups are of from 3 to 8 carbons.

The term an “effective amount” of a compound, as used herein, is thatamount sufficient to effect beneficial or desired results, such asclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that is an inhibitor of NOS, an effective amountof an agent is, for example, an amount sufficient to achieve a reductionin NOS activity as compared to the response obtained withoutadministration of the agent.

The terms “halogen” or “hal,” as used herein, represent bromine,chlorine, iodine, or fluorine.

The term “heteroaryl,” as used herein, represents that subset ofheterocycles, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system.

The terms “heterocycle” or “heterocyclyl,” as used interchangeablyherein represent a 5-, 6- or 7-membered ring, unless otherwisespecified, containing one, two, three, or four heteroatoms independentlyselected from the group consisting of nitrogen, oxygen and sulfur. The5-membered ring has zero to two double bonds and the 6- and 7-memberedrings have zero to three double bonds. The term “heterocyclyl” alsorepresents a heterocyclic compound having a bridged multicyclicstructure in which one or more carbons and/or heteroatoms bridges twonon-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group.The term “heterocycle” includes bicyclic, tricyclic and tetracyclicgroups in which any of the above heterocyclic rings is fused to one,two, or three rings, e.g., an aryl ring, a cyclohexane ring, acyclohexene ring, a cyclopentane ring, a cyclopentene ring and anothermonocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl,tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples offused heterocycles include tropanes and1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl,pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl,imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl,homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl,oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl,thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl,benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidinyl,isothiazolyl, isoindazoyl, triazolyl, tetrazolyl, oxadiazolyl, uricyl,thiadiazolyl, pyrimidyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl,tetrahydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl,benzofuranyl, benzothienyl and the like. Heterocyclic groups alsoinclude groups of the formula

where

F′ is selected from the group consisting of —CH₂—, —CH₂O— and —O—, andG′ is selected from the group consisting of —C(O)— and—(C(R′)(R″))_(v)—, where each of R′ and R″ is, independently, selectedfrom the group consisting of hydrogen or alkyl of one to four carbonatoms, and v is one to three and includes groups, such as1,3-benzodioxolyl, 1,4-benzodioxanyl, and the like. Any of theheterocycle groups mentioned herein may be optionally substituted withone, two, three, four or five substituents independently selected fromthe group consisting of: (1) alkanoyl of one to six carbon atoms; (2)alkyl of one to six carbon atoms; (3) alkoxy of one to six carbon atoms;(4) alkoxyalkyl, where the alkyl and alkylene groups are independentlyof one to six carbon atoms; (5) alkylsulfinyl of one to six carbonatoms; (6) alkylsulfinylalkyl, where the alkyl and alkylene groups areindependently of one to six carbon atoms; (7) alkylsulfonyl of one tosix carbon atoms; (8) alkylsulfonylalkyl, where the alkyl and alkylenegroups are independently of one to six carbon atoms; (9) aryl; (10)amino; (11) aminoalkyl of one to six carbon atoms; (12) heteroaryl; (13)alkaryl, where the alkylene group is of one to six carbon atoms; (14)aryloyl; (15) azido; (16) azidoalkyl of one to six carbon atoms; (17)carboxaldehyde; (18) (carboxaldehyde)alkyl, where the alkylene group isof one to six carbon atoms; (19) cycloalkyl of three to eight carbonatoms; (20) alkcycloalkyl, where the cycloalkyl group is of three toeight carbon atoms and the alkylene group is of one to ten carbon atoms;(21) halo; (22) haloalkyl of one to six carbon atoms; (23) heterocyclyl;(24) (heterocyclyl)oxy; (25) (heterocyclyl)oyl; (26) hydroxy; (27)hydroxyalkyl of one to six carbon atoms; (28) nitro; (29) nitroalkyl ofone to six carbon atoms; (30) N-protected amino; (31) N-protectedaminoalkyl, where the alkylene group is of one to six carbon atoms; (32)oxo; (33) thioalkoxy of one to six carbon atoms; (34) thioalkoxyalkyl,where the alkyl and alkylene groups are independently of one to sixcarbon atoms; (35) —(CH₂)_(q)CO₂R^(A), where q is an integer of fromzero to four, and R^(A) is selected from the group consisting of (a)alkyl, (b) aryl, (c) alkaryl, and (d) hydrogen where the alkylene groupis of one to six carbon atoms; (36) —(CH₂)_(q)CONR^(B)R^(C), where q isan integer of from zero to four and where R^(B) and R^(C) areindependently selected from the group consisting of (a) hydrogen, (b)alkyl, (c) aryl, and (d) alkaryl, where the alkylene group is of one tosix carbon atoms; (37) —(CH₂)_(q)SO₂R^(D), where q is an integer of fromzero to four and where R^(D) is selected from the group consisting of(a) alkyl, (b) aryl, and (c) alkaryl, where the alkylene group is of oneto six carbon atoms; (38) —(CH₂)_(q)SO₂NR^(E)R^(F), where q is aninteger of from zero to four and where each of R^(E) and R^(F) is,independently, selected from the group consisting of (a) hydrogen, (b)alkyl, (c) aryl, and (d) alkaryl, where the alkylene group is of one tosix carbon atoms; (39) —(CH₂)_(q)NR^(G)R^(H), where q is an integer offrom zero to four and where each of R^(G) and R^(H) is, independently,selected from the group consisting of (a) hydrogen; (b) an N-protectinggroup; (c) alkyl of one to six carbon atoms; (d) alkenyl of two to sixcarbon atoms; (e) alkynyl of two to six carbon atoms; (f) aryl; (g)alkaryl, where the alkylene group is of one to six carbon atoms; (h)cycloalkyl of three to eight carbon atoms; and (i) alkcycloalkyl, wherethe cycloalkyl group is of three to eight carbon atoms, and the alkylenegroup is of one to ten carbon atoms, wherein in one embodiment no twogroups are bound to the nitrogen atom through a carbonyl group or asulfonyl group; (40) thiol; (41) perfluoroalkyl; (42) perfluoroalkoxy;(43) aryloxy; (44) cycloalkoxy; (45) cycloalkylalkoxy; and (46)arylalkoxy.

The term “(heterocycle)oxy,” as used herein, represent a heterocyclegroup, as defined herein, attached to the parent molecular group throughan oxygen atom.

The term“(heterocycle)oyl,” as used herein, represent a heterocyclegroup, as defined herein, attached to the parent molecular group througha carbonyl group.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached an N-protecting group, asdefined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, Protective Groups in Organic Synthesis, 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aroyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl groups such asbenzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forminggroups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl,2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical.

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein (e.g., any ofCompounds (1)-(33) and compounds of Formula (I)), formulated with apharmaceutically acceptable excipient, and typically manufactured orsold with the approval of a governmental regulatory agency as part of atherapeutic regimen for the treatment of disease in a mammal.Pharmaceutical compositions can be formulated, for example, for oraladministration in unit dosage form (e.g., a tablet, capsule, caplet,gelcap, or syrup); for topical administration (e.g., as a cream, gel,lotion, or ointment); for intravenous administration (e.g., as a sterilesolution free of particulate emboli and in a solvent system suitable forintravenous use); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, refers anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, or waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc,titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term “pharmaceutically acceptable prodrugs” as used herein,represents those prodrugs of the compounds of the present inventionwhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and animals with undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention.

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention orseparately by reacting the free base group with a suitable organic acid.Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphersulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts andthe like. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamineand the like.

The terms “pharmaceutically acceptable solvate” or “solvate,” as usedherein, means a compound of the invention wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent is physiologically tolerable at the dosage administered. Forexample, solvates may be prepared by crystallization, recrystallization,or precipitation from a solution that includes organic solvents, water,or a mixture thereof. Examples of suitable solvents are ethanol, water(for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone(NMP), dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF),N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

The term “Ph” as used herein means phenyl.

The term “prevent,” as used herein, refers to prophylactic treatment ortreatment that prevents one or more symptoms or conditions of a disease,disorder, or conditions described herein (e.g., acute pain, chronicpain, inflammatory pain, neuropathic pain, severe pain, cancer pain,migraine (with or without aura or allodynia), or chronic tension typeheadache). Preventative treatment can be initiated, for example, priorto (“pre-exposure prophylaxis”) or following (“post-exposureprophylaxis”) an event that precedes the onset of the disease, disorder,or conditions (e.g., exposure to a migraine trigger, to another cause ofpain, or to a pathogen). Preventive treatment that includesadministration of a compound of the invention, or a pharmaceuticalcomposition thereof, can be acute, short-term, or chronic. The dosesadministered may be varied during the course of preventative treatment.See also: Kaniecki et al., “Treatment of Primary Headache: PreventiveTreatment of Migraine.” In: Standards of Care for Headache Diagnosis andTreatment. Chicago (IL): National Headache Foundation; 2004. p. 40-52.

The term “prodrug,” as used herein, represents compounds which arerapidly transformed in vivo to the parent compound of the above formula(e.g., compounds of Formula (I) and compounds (1)-(33)), for example, byhydrolysis in blood. Prodrugs of the compounds of the invention may beconventional esters. Some common esters which have been utilized asprodrugs are phenyl esters, aliphatic (C₇-C₈ or C₈-C₂₄) esters,cholesterol esters, acyloxymethyl esters, carbamates, and amino acidesters. For example, a compound of the invention that contains an OHgroup may be acylated at this position in its prodrug form. A thoroughdiscussion is provided in T. Higuchi and V. Stella, Pro-drugs as NovelDelivery Systems, Vol. 14 of the A.C.S. Symposium Series, Edward B.Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, and Judkins et al.,Synthetic Communications 26(23):4351-4367, 1996, each of which isincorporated herein by reference. Preferably, prodrugs of the compoundsof the present invention are pharmaceutically acceptable.

Each of the terms “selectively inhibits nNOS” or “a selective nNOSinhibitor” refers to a substance that inhibits or binds the nNOS isoformmore effectively than the eNOS and/or iNOS isoform as measured by an invitro assay, such as, for example, those assays described herein.Selective inhibition can be expressed in terms of an IC₅₀ value, a K_(i)value, or the inverse of a percent inhibition value which is lower, orconversely a higher % inhibition when the substance is tested in an nNOSassay than when tested in an eNOS and/or iNOS assay. Preferably, theIC₅₀ or K_(i) value is 2 times lower. More preferably, the IC₅₀ or K,value is 5, 10, 50, or even more than 100 times lower.

The term “spirocycle,” as used herein, represents an alkylene diradical,both ends of which are bonded to the same carbon atom of the parentgroup to form a spirocyclic group and also heteroalkylene diradical,both ends of which are bonded to the same atom.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a thioalkoxy groupsubstituted with an aryl group

The term “thioalkheterocyclyl,” as used herein, represents a thioalkoxygroup substituted with a heterocyclyl group.

The term “thioalkoxy,” as used herein, represents an alkyl groupattached to the parent molecular group through a sulfur atom. Exemplaryunsubstituted thioalkoxy groups are of from 1 to 6 carbons.

The term “thiol” represents an —SH group.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, such as clinicalresults. Beneficial or desired results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions;diminishment of extent of disease, disorder, or condition; stabilized(i.e. not worsening) state of disease, disorder, or condition;preventing spread of disease, disorder, or condition; delay or slowingthe progress of the disease, disorder, or condition; amelioration orpalliation of the disease, disorder, or condition; and remission(whether partial or total), whether detectable or undetectable.“Treatment” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment. “Palliating” a disease, disorder,or condition means that the extent and/or undesirable clinicalmanifestations of the disease, disorder, or condition are lessenedand/or time course of the progression is slowed or lengthened, ascompared to the extent or time course in the absence of treatment.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the protocol for testing thermal hyperalgesia in the Chungneuropathic pain model. The L5/L6 spinal nerve was surgically ligatedand animals allowed to recover for a period of 7-10 days. During thisperiod animals develop neuropathic pain. The reduction of paw withdrawallatency after an infrared thermal stimulus (post-SNL) was measuredfollowing the induction period for comparison with pre-surgery baselinelevels (BL). Following drug administration, thermal hyperalgesia wasmeasured at various time points.

FIG. 2 shows the protocol for testing mechanical allodynia in the Chungneuropathic pain model. The L5/L6 spinal nerve was surgically ligatedand animals allowed to recover for a period of 7-10 days. During thisperiod animals develop neuropathic pain. The reduction of tactilethresholds (post-SNL) was measured following the induction period forcomparison with pre-surgery baseline levels (BL). Following drugadministration, tactile allodynia was measured at various time pointswith calibrated von-Frey filaments.

FIG. 3 shows the reversal of thermal hyperalgesia in rats after i.p.administration of Compound (8) (30 mg/kg) in the L5/L6 spinal nerveligation model of neuropathic pain (Chung model).

FIG. 4 shows the effects of Compound (8) after i.p. administration (30mg/kg dose) on the reversal of tactile allodynia in rats after L5/L6spinal nerve ligation (Chung model).

FIG. 5 shows the antiallodynic effect of Compound (8) afteradministration (3 mg/kg p.o.) in the dural inflammation model ofmigraine.

FIG. 6 shows the reversal of mechanical allodynia followingadministration (60, 100, or 200 mg/kg, p.o.) of Compound (8) in theCarrageenan model of inflammatory pain.

FIG. 7 shows the reversal of thermal hyperalgesia followingadministration (60, 100, or 200 mg/kg, p.o.) of Compound (8) in theCarrageenan model of inflammatory pain.

DETAILED DESCRIPTION

The invention features novel benzoxazines, benzothiazines, and relatedcompounds having nitric oxide synthase (NOS) inhibitory activity,pharmaceutical and diagnostic compositions containing them, and theirmedical use. Exemplary compounds of the invention are shown in Table 2.

TABLE 2

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

Exemplary methods for synthesizing compounds of the invention aredescribed herein.

Methods of Preparing Compounds of the Invention

The compounds of the invention can be prepared by processes analogous tothose established in the art, for example, by the reaction sequencesshown in Schemes 1-5. The numbering system used for the general schemesdoes not necessarily correspond to that employed elsewhere in thedescription or in the claims.

A compound of formula C can be prepared under standard alkylatingconditions by treating a compound of formula A with a compound offormula B, or a suitably protected derivative thereof, and “LG” is aleaving group such as chloro, bromo, iodo, or sulfonate (e.g., mesylate,tosylate, or triflate). Conditions to effect the alkylation of acompound of formula A with a compound of formula B may include heating acompound of formula A and a compound of formula B with or without asolvent, preferably with a suitable solvent such as DMF, optionally inthe presence of a suitable base, such as potassium or sodium carbonateor sodium hydride (see Scheme 1).

A compound of formula D can be prepared by reduction of the nitro groupof a compound of formula C or a suitably protected derivative, understandard conditions as shown in Scheme 1. In one example, standardreduction conditions include the use of Raney Nickel in a polar solvent,such as methanol at refluxing temperatures. Alternatively, a compound offormula D can be prepared by the hydrogenation of a compound of formulaC using a suitable catalyst (e.g., palladium on charcoal) in ethanol, oranother solvent or combinations of solvents. As shown in Scheme 1, acompound of formula F can be prepared by reacting a compound of formulaE with a compound of formula D according to a previous procedure (U.S.Patent Publication No. 20060258721 A1, herein incorporated byreference).

Alternatively, compound of formula K in which R¹ is (CH₂)_(n)X¹, whereX¹ is

with R^(1A), R^(1B), R^(1C), R^(1D), Y¹ is CH₂, O, S, NR¹, n1, p1, andq1 as defined by example herein, involves the reaction of a compound offormula H, wherein LG is a suitable leaving group, such as chloro,bromo, iodo, or sulfonate (e.g., mesylate, tosylate, or triflate), withcompounds of formula I under standard alkylation conditions as shown inScheme 2. When LG is an aldehyde or ketone group, standard reductiveamination conditions (e.g., Abdel-Majid et al. J. Org. Chem.61:3849-3862, 1996) may be employed using a suitable reducing agent suchas NaBH₄, NaBH(OAc)₃, NaCNBH₄, and the like, in an alcoholic solvent,such as ethanol, to produce a compound of formula J. The reductiveamination may be performed in one reaction, or the imine resulting frommixing a compound of formula H with a compound of formula I can bepre-formed in situ, followed by sequential reduction with a suitablereducing agent. Compound J is converted to compound K by nitro reductionfollowed by amidation in a similar fashion as described in Scheme 1.

Compounds of general formula L can be prepared from a compound offormula D by amide reduction with lithium aluminum hydride in aproticsolvents (Scheme 3). Alternatively, a compound of formula L can bereduced using a suitable reducing agent, such as BH₃. These compoundsare then converted to compound of formula M by coupling with reagent Eas described in Scheme 1.

Compounds of general formula P can be prepared from compound N andcompound of general formula O under standard reductive aminationconditions (Scheme 4; Abdel-Majid et al. J. Org. Chem. 61:3849-3862,1996). Compounds of general formula R can be prepared by aromatichalogenation of compounds of general formula P according to establishedprocedures (see, for example, de la Mare, “Electrophilic Halogenation,”Cambridge University Press, Cambridge (1976)). The preferred conditionsinclude reacting compounds of general formula P with N-bromosuccinimideunder neutral conditions A compound of formula S can be prepared bymetal catalyzed amination of a compound of formula R where X is chloro,bromo, or iodo (Wolfe et al. J. Org. Chem. 65:1158-1174, 2000) in thepresence of a suitable ammonia equivalent, such as benzophenone imine,LiN(SiMe₃)₂, Ph₃SiNH₂, NaN(SiMe₃)₂, or lithium amide (Huang andBuchwald, Org. Lett. 3(21):3417-3419, 2001). A preferred halogen isbromo in the presence of palladium (0) or palladium (II) catalyst.Examples of suitable metal catalysts include, for example, a palladiumcatalyst coordinated to suitable ligands. Suitable ligands for palladiumcan vary greatly and may include4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (XantPhos),2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP),bis(2-diphenylphosphinophenyl)ether (DPEphos),1,1′-bis(diphenylphosphino)ferrocene (dppf),1,2-bis-diphenylphosphinobutane (dppb),1,3-bis(diphenylphosphino)propane (dppp), (o-biphenyl)-P(t-Bu)₂,(o-biphenyl)-P(Cy)₂, P(t-Bu)₃, P(Cy)₃, and others (e.g., Huang andBuchwald, Org. Lett. 3(21):3417-3419, 2001). Preferably the ligand isP(t-Bu)₃. The Pd-catalyzed amination is performed in a suitable solvent,such as THF, dioxane, toluene, xylene, DME, and the like, attemperatures between room temperature and reflux. Conversion of CompoundS to T was done under conditions in Scheme 1.

A compound of formula V, where hal=F, Cl, Br, or I, can be prepared fromcompound U and compound O (Scheme 5) under standard reductive aminationconditions as previously described in Scheme 4. When hal=F or Cl, acompound of formula Z can be prepared from a compound of formula V byreacting with W under basic conditions. A suitable base such as K₂CO₃ ina suitable solvent like DMF. In some cases, heat may be required forthis transformation. Alternatively, when hal=Cl, Br or I, compound Z canbe prepared from compound V and W by a transition metal catalyzedreaction. Preferred conditions involves the use of a palladium catalystsuch as Pd(OAc)₂ or Pd₂(dba)₃ with a phosphine ligand such as Josiphosor CyPF^(t)Bu (Hartwig, Acc. Chem. Res., 2008, 41, 1534). The hydroxylgroup of compound Z can be converted to a LG such as chloro, bromo,iodo, or sulfonate (e.g. mesylate, tosylate, or triflate) to givecompound of general formula Y by employing standard conditions. Thepreferred leaving group is iodo, and it can be prepared usingtriphenylphosphine and iodine in a suitable solvent such as THF.Compound Y can be cyclized to compound Z by heating with a base such asK₂CO₃ in a suitable solvent such as DMF. Compounds of formula Z can beconverted to compounds of the invention A1 by nitro reduction followedby amidine coupling as described previously in Scheme 1.

In some cases the chemistries outlined above may have to be modified,for instance, by the use of protective groups to prevent side reactionsdue to reactive groups, e.g., attached as substituents. This may beachieved by means of conventional protecting groups as described inProtective Groups in Organic Chemistry, McOmie, Ed., Plenum Press, 1973and in Greene and Wuts, Protective Groups in Organic Synthesis, JohnWiley & Sons, 3^(rd) Edition, 1999.

The compounds of the invention, and intermediates in the preparation ofthe compounds of the invention, may be isolated from their reactionmixtures and purified (if necessary) using conventional techniques,including extraction, chromatography, distillation, andrecrystallization.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid ina suitable solvent, and the formed salt is isolated by filtration,extraction, or any other suitable method.

The formation of solvates of the compounds of the invention will varydepending on the compound and the solvate. In general, solvates areformed by dissolving the compound in the appropriate solvent andisolating the solvate by cooling or adding an antisolvent. The solvateis typically dried or azeotroped under ambient conditions.

Preparation of an optical isomer of a compound of the invention may beperformed by reaction of the appropriate optically active startingmaterials under reaction conditions which will not cause racemization.Alternatively, the individual enantiomers may be isolated by separationof a racemic mixture using standard techniques, such as fractionalcrystallization or chiral HPLC.

A radiolabeled compound of the invention may be prepared using standardmethods known in the art. For example, tritium may be incorporated intoa compound of the invention using standard techniques, such ashydrogenation of a suitable precursor to a compound of the inventionusing tritium gas and a catalyst. Alternatively, a compound of theinvention containing radioactive iodine may be prepared from thecorresponding trialkyltin (preferably trimethyltin) derivative usingstandard iodination conditions, such as [¹²⁵I] sodium iodide in thepresence of chloramine-T in a suitable solvent, such asdimethylformamide. The trialkyltin compound may be prepared from thecorresponding non-radioactive halogen, preferably iodo, compound usingstandard palladium-catalyzed stannylation conditions, such as, forexample, hexamethylditin in the presence of tetrakis(triphenylphosphine)palladium (0) in an inert solvent, such as dioxane, and at elevatedtemperatures, preferably 50-100° C.

Pharmaceutical Uses

The present invention features all uses for compounds of the invention,including use in therapeutic methods, whether alone or in combinationwith another therapeutic substance, their use in compositions forinhibiting NOS activity, e.g., nNOS, their use in diagnostic assays, andtheir use as research tools.

The compounds of the invention have useful NOS inhibiting activity, andtherefore are useful for treating, or reducing the risk of, diseases orconditions that are ameliorated by a reduction in NOS activity. Suchdiseases or conditions include those in which the synthesis oroversynthesis of nitric oxide plays a contributory part.

Data substantiate the role of nitric oxide (NO) as a mediator ofneurotransmission, synaptic plasticity, and pathologic pain in thecentral and peripheral nervous systems (Snyder, Science 1992,257:494-496; Meller et al., Pain 52: 127-136, 1993; Praset et al., A.Prog. Neurobiol. 64: 51-68, 2001; and Choi et al., J. Neurol. Sci.138(1-2):14-20, 1996). Multiple studies in animal models and geneticknockouts suggest a pivotal role for NO and nNOS in the pathobiology ofsensitized pains such as neuropathic pain (Choi et al., J. Neurol. Sci.138(1-2):14-20, 1996) and the associated behavioral responses such asthermal hyperalgesia (Meller et al., Neurosci. 50:7-10, 1992; Yamamotoet al., Anesthesiology 82:1266-1273, 1995) and mechanical allodynia (Haoet al. Pain, 66:313-319, 1996; Pan et al., Anesthesiology 89(6):1518-23, 1998). For example, spinal nerve injury in mice leads to thedevelopment of mechanical hypersensitivity in wildtype but not nNOSknockout mice (Guan et al., Mol. Pain, 3:29, 2007). Similarly, systemicor intrathecally administered NOS inhibitors such as L-NAME or 7-NI canreduce nerve injury-induced mechanical hypersensitivity. This mechanicalhypersensitivity is accompanied by an increase in nNOS proteinexpression, but not eNOS or iNOS, in the ipsilateral L5 dorsal rootganglion 7-days post-nerve injury (Guan et al., Mol. Pain, 3:29, 2007).Gene expression studies have shown that NIDD, a protein regulating nNOSenzyme activity, is upregulated in the spinal cord and dorsal rootganglia (DRG) in a rat model of neuropathic or inflammatory pain (Chenet al. J. Mol. Histol. 39(2): 125-33, 2008). In the Chung model ofneuropathic pain (Spared Nerve Ligation or SNL), NOS inhibitors such asL-NAME can reduce neuropathic pain-like behavioral responses such asmechanical and cold allodynia, ongoing pain (Yoon et al., NeuroReport 9:367-372, 1998) and thermal hyperalgesia (see, for example, U.S. Pat. No.7,375,219 and PCT Publication No. WO 2009/062318, each of which ishereby incorporated by reference). NOS inhibitors have shown efficacy inother sensitized pain states with central sensitization components suchas vincristine (chemotherapy) induced painful neuropathy (Kamei et al.Pain. 117(1-2):112-20, 2005), Complete Freund's adjuvant (CFA)-inducedchronic inflammatory pain (Chun et al., Pain 119: 113-123, 2005),carrageenan-induced mechanical and thermal hyperalgesia (Handy et al.,Neuropharmacology 37:37-43, 1998; Osborne et al., Br. J. Pharmacol.126(8):1840-1846, 1999), visceral hyperalgesia after intracoloniczymosan instillation (Coutinho et al. Eur. J. Pharmacol. 429: 319-325,2001), and postherpetic allodynia (Sasaki et al. Neuroscience, 150(2):459-466, 2007).

Accordingly, the present invention features a method of treating,preventing, or reducing the risk of, a disease or condition caused byNOS activity that includes administering an effective amount of acompound of the invention to a cell or animal in need thereof. Suchdiseases or conditions include, for example, migraine headache (with orwithout aura), chronic tension type headache (CTTH), migraine withallodynia, medication overuse headache, cluster headache; neuropathicpain such as AIDS associated painful neuropathy, central post-strokepain (CPSP), chronic headache, diabetic neuropathy, chemotherapy inducedneuropathic pain (e.g., paclitaxel, cis-Platin, Doxorubicin etc.),postherpetic neuralgia, trigeminal neuralgia; chronic inflammatory painresulting from osteoarthritis, rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, undifferentiated spondyloarthropathy,reactive arthritis, visceral pain, neuroinflammation, medication-inducedhyperalgesia and/or allodynia, e.g., opioid-induced hyperalgesia ortriptan (5-HT_(1D/1B) agonists)-induced hyperalgesia/allodynia, acutepain, chronic pain, diabetic neuropathy, bone cancer pain, chemicaldependencies or addictions, e.g., drug addiction, cocaine addition,nicotine addition, metamphetamine-induced neurotoxicity, ethanoltolerance, dependence, or withdrawal, or morphine/opioid inducedtolerance, dependence, hyperalgesia, or withdrawal, CNS disordersincluding but not limited to, e.g., epilepsy, anxiety, depression (aloneor in combination), attention deficit hyperactivity disorder (ADHD),psychosis, or dementia, neurodegenerative diseases or nerve injury,e.g., acute spinal cord injury, AIDS associated dementia, Parkinson'sdisease, Alzheimer's disease, ALS, Huntington's disease, multiplesclerosis, neurotoxicity, or head trauma, cardiovascular relatedconditions, e.g., stroke, CABG associated neurological damage,HCAcardiogenic shock, reperfusion injury, or vascular dementia, orgastrointestinal disorders, e.g., ileostomy-associated diarrhea, ordumping syndrome.

The following description is a summary and a basis for the link betweenNOS inhibition (particularly nNOS or nNOS and iNOS) and some of theseconditions.

Migraine with or without Aura

The first observation by Asciano Sobrero in 1847 that small quantitiesof nitroglycerine, an NO releasing agent, causes severe headache lead tothe nitric oxide hypothesis of migraine (Olesen et al., Cephalagia15:94-100, 1995). Serotonergic 5HT_(1D/1B) agonists, such assumatriptan, which are used clinically in the treatment of migraine, areknown to prevent the cortical spreading depression in the lissencephalicand gyrencephalic brain during migraine attack, a process resulting inwidespread release of NO. Indeed, it has been shown that sumatriptanmodifies the artificially enhanced cortical NO levels following infusionof glyceryl trinitrate in rats (Read et al., Brain Res. 847:1-8, 1999;ibid, 870(1-2):44-53, 2000). In a human randomized double-blindedclinical trial for migraine, a 67% response rate after single i.v.administration of L-N^(G) methylarginine hydrochloride (L-NMMA, an NOSinhibitor) was observed. The effect was not attributed to a simple eNOSmediated vasoconstriction since no effect was observed on transcranialdoppler determined velocity in the middle cerebral artery (Lassen etal., Lancet 349:401-402, 1997). In a recent adaptive clinical trialdesign for the treatment of acute migraine, administration of theselective iNOS inhibitor GW274150 at doses ranging from 5 to 180 mg wasno different than placebo in terms of the proportion of subjects whobecame pain free at two hours after treatment. The same compoundevaluated in a clinical trial of migraine prophylaxis (120 mg daily for12 weeks) was also ineffective in reducing the frequency of migraineattack (Hoye et al. Cephalalgia, 2009, 29, 132). However, in an openpilot study using the NO scavenger hydroxycobalamin, a reduction in thefrequency of migraine attack of 50% was observed in 53% of the patientsand a reduction in the total duration of migraine attacks was alsoobserved (van der Kuy et al., Cephalgia 22(7):513-519, 2002). Theresults from these clinical trials suggest that iNOS or eNOS do not playa significant role in the generation of migraine headache but ratherpoints to the nNOS as the key isoform in migraine headache. Thecompounds of the invention (e.g., a compound of Formula (I) such asCompounds (1)-(33)) may also be employed for migraine prophylaxis.

Migraine with Allodynia

Clinical studies have shown that as many as 75% of patients developcutaneous allodynia (exaggerated skin sensitivity) during migraineattacks and that its development during migraine is detrimental to theanti-migraine action of triptan 5HT_(1B/1D) agonists (Burstein et al.,Ann. Neurol. 47:614-624, 2000; Burstein et al., Brain, 123:1703-1709,2000). While the early administration of triptans such as sumatriptancan terminate migraine pain, late sumatriptan intervention is unable toterminate migraine pain or reverse the exaggerated skin sensitivity inmigraine patients already associated with allodynia (Burstein et al.,Ann. Neurol. DOI: 10.1002/ana.10785, 2003; Burstein and Jakubowski, Ann.Neurol., 55:27-36, 2004). The development of peripheral and centralsensitization correlates with the clinical manifestations of migraine.In migraine patients, throbbing occurs 5-20 minutes after the onset ofheadache, whereas cutaneous allodynia starts between 20-120 minutes(Burstein et al., Brain, 123:1703-1709, 2000). In the rat,experimentally induced peripheral sensitization of meningeal nociceptorsoccurs within 5-20 minutes after applying an inflammatory soup (I.S.) tothe dura (Levy and Strassman, J. Physiol., 538:483-493, 2002), whereascentral sensitization of trigeminovascular neurons develops between20-120 minutes (Burstein et al., J. Neurophysiol. 79:964-982, 1998)after I.S. administration. Parallel effects on the early or lateadministration of antimigraine triptans like sumatriptan on thedevelopment of central sensitization have been demonstrated in the rat(Burstein and Jakubowski, vide supra). Thus, early but not latesumatriptan prevents the long-term increase in I.S.-induced spontaneousactivity seen in central trigeminovascular neurons (a clinical correlateof migraine pain intensity). In addition, late sumatriptan interventionin rats did not prevent I.S.-induced neuronal sensitivity to mechanicalstimulation at the periorbital skin, nor decreased the threshold to heat(a clinical correlate of patients with mechanical and thermal allodyniain the periorbital area). In contrast, early sumatriptan prevented I.S.from inducing both thermal and mechanical hypersensitivity. After thedevelopment of central sensitization, late sumatriptan interventionreverses the enlargement of dural receptive fields and increases insensitivity to dural indentation (a clinical correlate of pain throbbingexacerbated by bending over) while early intervention prevents itsdevelopment.

Previous studies on migraine compounds such as sumatriptan (Kaube etal., Br. J. Pharmacol. 109:788-792, 1993), zolmitriptan (Goadsby et al.,Pain 67:355-359, 1996), naratriptan (Goadsby et al., Br. J. Pharmacol.,328:37-40, 1997), rizatriptan (Cumberbatch et al., Eur. J. Pharmacol.,362:43-46, 1998), or L-471-604 (Cumberbatch et al., Br. J. Pharmacol.126:1478-1486, 1999) examined their effects on nonsensitized centraltrigeminovascular neurons (under normal conditions) and thus do notreflect on their effects under the pathophysiological conditions ofmigraine. While triptans are effective in terminating the throbbing ofmigraine whether administered early or late, the peripheral action ofsumatriptan is unable to terminate migraine pain with allodyniafollowing late intervention via the effects of central sensitization oftrigeminovascular neurons. The limitations of triptans suggest thatimprovement in the treatment of migraine pain can be achieved byutilizing drugs that can abort ongoing central sensitization, such asthe compounds of the present invention.

It has been shown that systemic nitroglycerin increases nNOS levels andc-Fos-immunoreactive neurons (a marker neuronal activation) in rattrigeminal nucleus caudalis after 4 hours, suggesting NO likely mediatescentral sensitization of trigeminal neurons (Pardutz et al., Neuroreport11(14):3071-3075, 2000). In addition, L-NAME can attenuate Fosexpression in the trigeminal nucleus caudalis after prolonged (2 hours)electrical stimulation of the superior sagittal sinus (Hoskin et al.Neurosci. Lett. 266(3):173-6, 1999). Taken together with ability of NOSinhibitors to abort acute migraine attack (Lassen et al., Cephalalgia18(1):27-32, 1998), the compounds of the invention, alone or incombination with other antinociceptive agents, represent excellentcandidate therapeutics for aborting migraine in patients after thedevelopment of allodynia.

Chronic Headache (CTTH)

NO contributes to the sensory transmission in the peripheral (Aley etal., J. Neurosci. 1:7008-7014, 1998) and central nervous system (Mellerand Gebhart, Pain 52:127-136, 1993). Substantial experimental evidenceindicates that central sensitization, generated by prolonged nociceptiveinput from the periphery, increases excitability of neurons in the CNSand is caused by, or associated with, an increase in NOS activation andNO synthesis (Bendtsen, Cephalagia 20:486-508, 2000; Woolf and Salter,Science 288:1765-1769, 2000). It has been shown that experimentalinfusion of the NO donor, glyceryl trinitrate, induces headache inpatients. In a double-blinded study, patients with chronic tension-typeheadache receiving L-NMMA (an NOS inhibitor) had a significant reductionin headache intensity (Ashina and Bendtsen, J. Headache Pain 2:21-24,2001; Ashina et al., Lancet 243(9149):287-9, 1999). Thus the NOSinhibitors of the present invention may be useful for the treatment ofchronic tension-type headache.

Acute Spinal Cord Injury and Chronic or Neuropathic Pain

In humans, NO evokes pain on intracutaneous injection (Holthusen andArndt, Neurosci. Lett. 165:71-74, 1994), thus showing a directinvolvement of NO in pain. Furthermore, NOS inhibitors have little or noeffect on nociceptive transmission under normal conditions (Meller andGebhart, Pain 52:127-136, 1993). NO is involved in the transmission andmodulation of nociceptive information at the periphery, spinal cord andsupraspinal level (Duarte et al., Eur. J. Pharmacol. 217:225-227, 1992;Haley et al., Neuroscience 31:251-258, 1992). Lesions or dysfunctions inthe CNS may lead to the development of chronic pain symptoms, known ascentral pain, and includes spontaneous pain, hyperalgesia, andmechanical and cold allodynia (Pagni, Textbook of Pain, ChurchillLivingstone, Edinburgh, 1989, pp. 634-655; Tasker In: The Management ofPain, pp. 264-283, J. J. Bonica (Ed.), Lea and Febiger, Philadelphia,Pa., 1990; Casey, Pain and Central Nervous System Disease: The CentralPain Syndromes, pp. 1-11 K. L. Casey (Ed.), Raven Press, New York,1991). It has been demonstrated that systemic administration (i.p.) ofthe NOS inhibitors 7-NI and L-NAME relieve chronic allodynia-likesymptoms in rats with spinal cord injury (Hao and Xu, Pain 66:313-319,1996). The effects of 7-NI were not associated with a significantsedative effect and were reversed by L-arginine (NO precursor). Themaintenance of thermal hyperalgesia is believed to be mediated by nitricoxide in the lumbar spinal cord and can be blocked by intrathecaladministration of a nitric oxide synthase inhibitor like L-NAME orsoluble guanylate cyclase inhibitor methylene blue (Neuroscience50(1):7-10, 1992).

Neuropathic pain may be due to a primary insult to the peripheral (e.g.,trigeminal neuraligia) or central nervous system (e.g., thalamic pain).It is likely, however, that the pathphysiology leading to a neuropathicpain state originating from a peripheral lesion spreads over time toother areas to affect higher order neurons in the dorsal root ganglionor to alter descending inhibition of spinal pain transmission by braincenters such as periaqueductal gray, locus corealis (Zimmermann, Eur. J.Pharmacol. 429:23-27, 2001) or alter descending facilitory pathways inthe rostroventromedial medulla (RVM) (Bee and Dickenson, Pain, 140(1):209-23, 2008).

Thus the NOS inhibitors of the present invention may be useful for thetreatment of chronic or neuropathic pain.

Diabetic Neuropathy

The endogenous polyamine metabolite agmatine is a metabolite of argininethat is both an NOS inhibitor and N-methyl-D-aspartate (NMDA) channelantagonist. Agmatine is effective in both the spinal nerve ligation(SNL) model of neuropathic pain as well as the streptozotocin model ofdiabetic neuropathy (Karadag et al., Neurosci. Lett. 339(1):88-90,2003). Thus compounds possessing NOS inhibitory activity, such as, forexample, a compound of formula I, a combination of an NOS inhibitor andan NMDA antagonist should be effective in treating diabetic neuropathyand other neuropathic pain conditions.

Inflammatory Diseases and Neuroinflammation

LPS, a well-known pharmacological tool, induces inflammation in manytissues and activates NFκB in all brain regions when administeredintravenously. It also activates pro-inflammatory genes when injectedlocally into the striatum (Stern et al., J. Neuroimmunology,109:245-260, 2000). Recently it has been shown that both the NMDAreceptor antagonist MK801 and the brain selective nNOS inhibitor 7-NIboth reduce NFκB activation in the brain and thus reveal a clear rolefor glutamate and NO pathway in neuroinflammation (Glezer et al.,Neuropharmacology 45(8):1120-1129, 2003). Thus, the administration of acompound of the invention, either alone or in combination with an NMDAantagonist, should be effective in treating diseases arising fromneuroinflammation.

Stroke and Reperfusion Injury

The role of NO in cerebral ischemia can be protective or destructivedepending on the stage of evolution of the ischemic process and on thecellular compartment producing NO (Dalkara et al., Brain Pathology 4:49,1994). While the NO produced by eNOS is likely beneficial by acting as avasodilator to improve blood flow to the affected area (Huang et al., J.Cereb. Blood Flow Metab. 16:981, 1996), NO produced by nNOS contributesto the initial metabolic deterioration of the ischemic penumbra,resulting in larger infarcts (Hara et al., J. Cereb. Blood Flow Metab.16:605, 1996). The metabolic derangement that occurs during ischemia andsubsequent reperfusion results in the expression and release of severalcytokines that activate iNOS in several cell types including some of thecentral nervous system. NO can be produced at cytotoxic levels by iNOS,and increased levels of iNOS contribute to progressive tissue damage inthe penumbra, leading to larger infarcts (Parmentier et al., Br. J.Pharmacol. 127:546, 1999). Inhibition of i-NOS has been shown toameliorate cerebral ischemic damage in rats (Am. J. Physiol. 268:R286,1995).

It has been shown that a synergistic neuroprotective effect is observedupon the combined administration of an NMDA antagonist (e.g., MK-801 orLY293558) with nNOS selective inhibitors (7-NI or ARL17477) in globalcerebral ischemia (Hicks et al., Eur. J. Pharmacol. 381:113-119, 1999).Thus the compounds of the invention, administered either alone or incombination with NMDA antagonists, or compounds possessing mixednNOS/NMDA activity, may be effective in treating conditions of strokeand other neurodegenerative disorders.

Complications Resulting from Coronary Artery Bypass Surgery

Cerebral damage and cognitive dysfunction still remains as a majorcomplication of patients undergoing coronary artery bypass surgery(CABG) (Roch et al., N. Eng. J. Med. 335:1857-1864, 1996; Shaw et al.,Q. J. Med. 58:59-68, 1986). This cerebral impairment following surgeryis a result of ischemia from preoperative cerebral microembolism. In arandomized trial of the NMDA antagonist remacemide, patients showed asignificant overall postoperative improvement in learning ability inaddition to reduced deficits (Arrowsmith et al., Stroke 29:2357-2362,1998). Given the involvement of excitotoxicity produced by excessiverelease of glutamate and calcium influx, it is expected that aneuroprotective agent, such as a compound of the invention or an NMDAantagonist, either alone or in combination (as discussed above), mayhave a beneficial effect improving neurological outcomes after CABG.

AIDS-Associated Dementia

HIV-1 infection can give rise to dementia. The HIV-1 coat protein gp-120kills neurons in primary cortical cultures at low picomolar levels andrequires external glutamate and calcium (Dawson et al., 90(8):3256-3259,1993). This toxicity can be attenuated by administration of aneuroprotective agent, e.g., a compound of the invention, either aloneor in combination with another therapeutic agent, such as, for example,an NMDA antagonist (as discussed above).

Examples of other compounds, e.g., NMDA antagonists, useful for any ofthe combinations of the invention include aptiganel; besonprodil;budipine; conantokin G; delucemine; dexanabinol; felbamate;fluorofelbamate; gacyclidine; glycine; ipenoxazone; kaitocephalin;lanicemine; licostinel; midafotel; milnacipran; neramexane;orphenadrine; remacemide; topiramate;(αR)-α-amino-5-chloro-1-(phosphonomethyl)-1H-benzimidazole-2-propanoicacid; 1-aminocyclopentane-carboxylic acid;[5-(aminomethyl)-2-[[[(5S)-9-chloro-2,3,6,7-tetrahydro-2,3-dioxo-1H-,5H-pyrido[1,2,3-de]quinoxalin-5-yl]acetyl]amino]phenoxy]-aceticacid; α-amino-2-(2-phosphonoethyl)-cyclohexanepropanoic acid;α-amino-4-(phosphonomethyl)-benzeneacetic acid;(3E)-2-amino-4-(phosphonomethyl)-3-heptenoic acid;3-[(1E)-2-carboxy-2-phenylethenyl]-4,6-dichloro-1H-indole-2-carboxylicacid; 8-chloro-2,3-dihydropyridazino[4,5-b]quinoline-1,4-dione 5-oxidesalt with 2-hydroxy-N,N,N-trimethyl-ethanaminium;N′-[2-chloro-5-(methylthio)phenyl]-N-methyl-N-[3-(methylthio)phenyl]-guanidine;N′-[2-chloro-5-(methylthio)phenyl]-N-methyl-N-[3-[(R)-methylsulfinyl]phenyl]-guanidine;6-chloro-2,3,4,9-tetrahydro-9-methyl-2,3-dioxo-1H-indeno[1,2-b]pyrazine-9-aceticacid; 7-chlorothiokynurenic acid;(3S,4aR,6S,8aR)-decahydro-6-(phosphonomethyl)-3-isoquinolinecarboxylicacid;(−)-6,7-dichloro-1,4-dihydro-5-[3-(methoxymethyl)-5-(3-pyridinyl)-4-H-1,2,4-triazol-4-yl]-2,3-quinoxalinedione;4,6-dichloro-3-[(E)-(2-oxo-1-phenyl-3-pyrrolidinylidene)methyl]-1H-indole-2-carboxylicacid;(2R,4S)-rel-5,7-dichloro-1,2,3,4-tetrahydro-4-[[(phenylamino)carbonyl]amino]-2-quinolinecarboxylicacid;(3R,4S)-rel-3,4-dihydro-3-[4-hydroxy-4-(phenylmethyl)-1-piperidinyl-]-2H-1-benzopyran-4,7-diol;2-[(2,3-dihydro-1H-inden-2-yl)amino]-acetamide;1,4-dihydro-6-methyl-5-[(methylamino)methyl]-7-nitro-2,3-quinoxalinedione;[2-(8,9-dioxo-2,6-diazabicyclo[5.2.0]non-1(7)-en-2-yl)ethyl]-phosphonicacid;(2R,6S)-1,2,3,4,5,6-hexahydro-3-[(2S)-2-methoxypropyl]-6,11,11-trimethyl-2,6-methano-3-benzazocin-9-ol;2-hydroxy-5-[[(pentafluorophenyl)methyl]amino]-benzoic acid;1-[2-(4-hydroxyphenoxy)ethyl]-4-[(4-methylphenyl)methyl]-4-piperidinol;1-[4-(1H-imidazol-4-yl)-3-butynyl]-4-(phenylmethyl)-piperidine;2-methyl-6-(phenylethynyl)-pyridine;3-(phosphonomethyl)-L-phenylalanine; and3,6,7-tetrahydro-2,3-dioxo-N-phenyl-1H,5H-pyrido[1,2,3-de]quinoxaline-5-acetamideor those described in U.S. Pat. Nos. 6,071,966; 6,034,134; and5,061,703.

Cardiogenic Shock

Cardiogenic shock (CS) is the leading cause of death for patients withacute myocardial infarction that is consistent with increased levels ofNO and inflammatory cytokines. High levels of NO and peroxynitrite havemany effects, including a direct inhibition on myocardialcontractability, suppression of mitochondrial respiration in myocardium,alteration in glucose metabolism, reduced catecholamine responsivity,and induction of systemic vasodilation (Hochman, Circulation 107:2998,2003). In a clinical study in 11 patients with persistent shock,administration of the NOS inhibitor L-NMMA resulted in increases inurine output and blood pressure and survival rate of 72% up to 30 days(Cotter et al., Circulation 101:1258-1361, 2000). In a randomized trialof 30 patients, it was reported that L-NAME reduced patient mortalityfrom 67% to 27% (Cotter et al., Eur. Heart. J. 24(14):1287-95, 2003).Similarly, administration of a compound of the invention, either aloneor in combination with another therapeutic agent, may be useful for thetreatment of cardiogenic shock.

Anxiety and Depression

Recent studies of rats and mice in the forced swimming test (FST)indicate that NOS inhibitors have antidepressant activity in mice(Harkin et al. Eur. J. Pharm. 372:207-213, 1999) and that their effectis mediated by a serotonin dependent mechanism (Harkin et al.,Neuropharmacology 44(5):616-623, 1993). 7-NI demonstrates anxiolyticactivity in the rat plus-maze test (Yildiz et al., Pharmacology,Biochemistry and Behavior 65:199-202, 2000), whereas the selective nNOSinhibitor TRIM is effective in both the FST model of depression andanxiety in the light-dark compartment test (Volke et al., BehavioralBrain Research 140(1-2):141-7, 2003). Administration of a compound ofthe invention to an afflicted individual, either alone or in combinationwith another therapeutic agent, such as, for example, an antidepressant,may be useful for the treatment of anxiety or depression.

Attention Deficit Hyperactivity Disorder

Non-selective attention (NSA) to environmental stimuli in SpontaneouslyHypertensive (SHR) and Naples Low-Excitability (NHE) rats has been usedas an animal model of Attention-Deficit Hyperactivity Disorder (ADHD)(Aspide et al., Behav. Brain Res. 95(1):23-33, 1998). These geneticallyaltered animals show increased episodes of rearing that have a shorterduration than observed in normal animals. A single injection of L-NAMEat 10 mg/kg produced an increase in rearing duration. Similarly, usingthe more neuronally selective 7-NINA, an increase in the rearingduration was observed after rapid administration (i.p.), while a slowrelease single release dose or a slow multiple release dose (s.c. inDMSO) resulted in the opposite effect. Thus, administration of acompound of the invention may be useful for the treatment of ADHD.

Psychosis

Phencyclidine (PCP) is a non-competitive NMDA channel blocker thatproduces behavioral side effects in human and mammals consistent withthose observed in patients with psychosis. In two animal models ofpsychosis, the nNOS selective inhibitor AR-R17477 antagonizedPCP-induced hyperlocomotion and PCP-induced deficit in prepulseinhibition of the acoustic response startle (Johansson et al.,Pharmacol. Toxicol. 84(5):226-33, 1999). These results suggest theinvolvement of nNOS in psychosis. Therefore, administration of acompound of the invention to an afflicted individual may be useful forthe treatment of this or related diseases or disorders.

Head Trauma

The mechanism of neurological damage in patients with head traumaparallels that of stroke and is related to excitotoxic calcium influxfrom excessive glutamate release, oxidative stress and free radicalproduction from mitochondrial dysfunction and inflammation (Drug &Market Development 9(3):60-63, 1998). Animals treated with nitric oxidesynthase (NOS) inhibitors, such as 7-NI and 3-bromo-7-nitroindazole,have shown an improvement in neurological deficits after experimentaltraumatic brain injury (TBI) (Mesenge et al., J. Neurotrauma 13:209-14,1996). Administration of a compound of the invention to an afflictedindividual may also be useful for the treatment of neurological damagein head trauma injuries.

Hypothermic Cardiac Arrest

Hypothermic cardiac arrest (HCA) is a technique used to protect fromischemic damage during cardiac surgery when the brain is sensitive todamage during the period of blood flow interruption. Variousneuroprotective agents have been used as adjunct agents during HCA andreducing nitric oxide production during HCA is predicted to result inimprovements in neurological function. This is based on previous studiesthat showed glutamate excitotoxicity plays a role in HCA-inducedneurologic damage (Redmond et al., J. Thorac. Cardiovasc. Surg.107:776-87, 1994; Redmond et al., Ann. Thorac. Surg. 59:579-84, 1995)and that NO mediates glutamate excitotoxicity (Dawson and Snyder, J.Neurosci. 14:5147-59, 1994). In a study of 32 dogs undergoing 2 hours ofHCA at 18° C., a neuronal NOS inhibitor was shown to reduce cerebral NOproduction, significantly reduce neuronal necrosis, and resulted insuperior neurologic function relative to controls (Tseng et al., Ann.Thorac. Surg. 67:65-71, 1999). Administration of a compound of theinvention may also be useful for protecting patients from ischemicdamage during cardiac surgery.

Neurotoxicity and Neurodegenerative Diseases

Mitochondrial dysfunction, glutamate excitotoxicity, and free radicalinduced oxidative damage appear to be the underlying pathogenesis ofmany neurodegenerative diseases, including amyotrophic lateral sclerosis(ALS), Parkinson's disease (PD), Alzheimer's disease (AD), andHuntington's disease (HD) (Schulz et al., Mol. Cell. Biochem.174(1-2):193-197, 1997; Beal, Ann. Neurol. 38:357-366, 1995), and NO isa primary mediator in these mechanisms. For example, it was shown byDawson et al., in PNAS 88(14):6368-6371, 1991, that NOS inhibitors like7-NI and L-NAME prevent neurotoxicity elicited by N-methyl-D-aspartateand related excitatory amino acids.

(a) Parkinson's Disease

Studies have also shown that NO plays an important role in1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity, acommonly used animal model of Parkinson's disease (Matthews et al.,Neurobiology of Disease 4:114-121, 1997). MPTP is converted to MPP+ byMAO-B and is rapidly taken up by the dopamine transporter into themitochondria of dopamine containing neurons with subsequent activationof nNOS resulting in neuronal death. Mutant mice lacking the nNOS gene,but not the eNOS gene, have reduced lesions in the substantia nigraafter MPP+ injection into the striatum. In primate studies, 7-NI exertsa profound neuroprotective and antiparkinsonium effect after MPTPchallenge (Hantraye et al., Nature Med. 2:1017-1021, 1996) as did thenon-specific inhibitor L-NAME (T. S. Smith et. al. Neuroreport 1994, 5,2598-2600). These results suggest that administration of an appropriatedose of an NOS inhibitor, such as, for example, a compound of theinvention, can be beneficial in the treatment of Parkinson's Disease.

(b) Alzheimer's Disease (AD)

The pathology of AD is associated with β-amyloid plaques infiltratedwith activated microglia and astrocytes. When cultured rat microglia areexposed to beta-amyloid, there is a prominent microglial release ofnitric oxide, especially in the presence of gamma-interferon (Goodwin etal., Brain Research 692(1-2):207-14, 1995). In cortical neuronalcultures, treatment with nitric oxide synthase inhibitors providesneuroprotection against toxicity elicited by human beta-amyloid (Resinket al., Neurosci. Abstr. 21:1010, 1995). Consistent with the glutamatehypothesis of excitoxicity in neurodegerative disorders, the weak NMDAantagonist amantadine increases the life expectancy of PD patients(Uitti et al., Neurology 46(6):1551-6, 1996). In a preliminary,placebo-controlled study of patients with vascular- or Alzheimer's-typedementia, the NMDA antagonist memantine was associated with improvedClinical Global Impression of Change and Behavioral Rating Scale forGeriatric Patients scores (Winblad and Poritis, Int. J. Geriatr.Psychiatry 14:135-46, 1999). These results suggest that administrationof an appropriate dose of an NOS inhibitor, such as, for example, acompound of the invention, can be beneficial in the treatment of AD.

(c) Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative diseasecharacterized by selective motor neuronal death. Accumulating evidencesuggests that the pathogenesis of ALS is the insufficient clearance ofglutamate through the glutamate transporter, and the specificdistribution of Ca²⁺-permeable AMPA receptors in spinal motor neurons,indicates a glutamate-induced neurotoxicity. Increased nNOSimmunoreactivity is found in the spinal cords (Sasaki et al., ActaNeuropathol. (Berl) 101(4):351-7, 2001) and glial cells (Anneser et al.,Exp. Neurol. 171(2):418-21, 2001) of ALS patients, implicating NO as animportant factor in the pathogenesis of ALS. These results suggest thatadministration of an appropriate dose of an NOS inhibitor, such as, forexample, a compound of the invention, can be beneficial in the treatmentof ALS.

(d) Huntington's Disease

The pathogenesis of Huntington's disease (HD) arising from a mutation inthe Htt protein is linked to excitotoxicity, oxidative stress andapoptosis, in all of which excessive NO has a clear role (Peterson etal., Exp. Neurol. 157:1-18, 1999). Oxidative damage is one of the majorconsequences of defects in energy metabolism and is present in HD modelsafter injection of excitotoxins and mitochondrial inhibitors (A.Petersen et. al., Exp. Neurol. 157:1-18, 1999). This mitochrondrialdysfunction is associated with the selective and progressive neuronalloss in HD (Brown et al., Ann. Neurol. 41:646-653, 1997). NO candirectly impair the mitochondrial respiratory chain complex IV(Calabrese et al., Neurochem. Res. 25:1215-41, 2000). Striatal mediumspiny neurons appear to be the primary target for the generation ofmotor dysfunction in HD. Hyperphosphorylation and activation of NMDAreceptors on these neurons likely participates in the generation ofmotor dysfunction. It has been shown clinically that the NMDA antagonistamantadine improves choreiform dyskinesias in HD (Verhagen Metman etal., Neurology 59:694-699, 2002). Given the role of nNOS in NMDAmediated neurotoxicity, it is expected that nNOS inhibitors, especiallythose with mixed nNOS/NMDA, or combinations of drugs with nNOS and NMDAactivity will also be useful in ameliorating the effects and orprogression of HD. For example, pretreatment of rats with7-nitroindazole attenuates the striatal lesions elicited by stereotaxicinjections of malonate, an injury that leads to a condition resemblingHuntington's disease (Hobbs et. al., Ann. Rev. Pharm. Tox. 39:191-220,1999). In a R6/1 transgenic mouse model of HD expressing a human mutatedhtt exon1, a 116 CAG repeat, mice at 11, 19 and 35 weeks show aprogressive increase in lipid peroxidation with normal levels ofsuperoxide dismutase (SOD) at 11 weeks similar to wild type (WT) mice; amaximum level at 19 weeks, above that observed in WT mice andcorresponding to the early phase of disease progression; and finally,decreasing levels at 35 weeks below that observed in WT mice(Pérez-Sevriano et al., Brain Res. 951:36-42, 2002). The increase in SODactivity is attributable to a compensatory neuroprotective mechanism,with decreased levels at 35 weeks corresponding to a failed protectivemechanism. Concomitant with the levels of SOD, levels of calciumdependent NOS was the same for 11 week mice in both WT and R6/1 mice,but increased significantly at 19 weeks and decreased at 35 weeksrelative to WT control mice. Levels of nNOS expression also increaseddramatically relative to controls at 19 weeks but were decreasedsignificantly relative to controls at 35 weeks. No significantdifferences were observed in levels of eNOS expression, nor could iNOSprotein be detected during progression of the disease. The progressivephenotypic expression of the disease, as measured by increased weightloss, feet clasping behavior, and horizontal and vertical movements, areconsistent with changes in NOS activity and nNOS expression. Finally,the effects of L-NAME administration to both R6/2 transgenic HD mice andWT mice showed improved levels of clasping behavior at a 10 mg/kg dosesimilar to controls, which worsened at the highest dose of 500 mg/kg(Deckel et al., Brain Res. 919 (1):70-81, 2001). An improvement inweight increase in HD mice was also significant at the 10 mg/kg dose,but decreased relative to controls at high dose levels of L-NAME. Theseresults demonstrate that administration of an appropriate dose of an NOSinhibitor, such as, for example, a compound of the invention, can bebeneficial in the treatment of HD.

(e) Multiple Sclerosis (MS)

MS is in an inflammatory demyelinating disease of the CNS involvingcytokines and other inflammatory mediators. Many studies suggest that NOand its reactive derivative peroxynitrite are implicated in thepathogenesis of MS (Acar et al. J. Neurol. 250(5):588-92, 2003;Calabrese et al., Neurochem. Res. 28(9):1321-8, 2003). In experimentalautoimmune encephalomyelitis (EAE), a model of MS, nNOS levels areslightly increased in the spinal cord of EAE rats and treatment with7-nitroindazole results in a significant delay in the onset of EAEparalysis (Shin, J. Vet. Sci. 2(3):195-9, 2001). These results suggestthat administration of an appropriate dose of an NOS inhibitor, such as,for example, a compound of the invention, can be beneficial in thetreatment of MS.

(f) Methamphetamine-Induced Neurotoxicity

Methamphetamine is neurotoxic by destroying dopamine nerve terminals invivo. It has been shown that methamphetamine-induced neurotoxicity canbe attenuated by treatment with NOS inhibitors in vitro (Sheng et al.,Ann. N.Y. Acad. Sci. 801:174-186, 1996) and in in vivo animal models(Itzhak et al., Neuroreport 11(13):2943-6, 2000). Similarly, the nNOSselective inhibitor AR-17477AR, at 5 mg/kg s.c in mice, was able toprevent the methamphetamine-induced loss of the neurofilament proteinNF68 in mouse brain and prevent the loss of striatal dopamine andhomovanillic acid (HVA) (Sanchez et al., J. Neurochem. 85(2):515-524,2003). These results suggest that administration of an appropriate doseof an NOS inhibitor, such as, for example, a compound of the invention,can be beneficial in the treatment of methamphetamine-inducedneurotoxicity.

Administration of a compound of the invention, either alone or incombination with another therapeutic agent, such as, for example, anNMDA antagonist, may be useful for the protection or treatment of any ofthe neurodegenerative diseases described herein. Further, the compoundsof the invention may be tested in standard assays used to assessneuroprotection (see for example, Am. J. Physiol. 268:R286, 1995).

Chemical Dependencies and Drug Addictions (e.g., Dependencies on Drugs,Alcohol and Nicotine)

A key step in the process of drug-induced reward and dependence is theregulation of dopamine release from mesolimbic dopaminergic neurons.Chronic application of cocaine alters the expression of the key proteincontrolling the synaptic level of dopamine—the dopamine transporter(DAT).

(a) Cocaine Addiction

Studies have shown that animals reliably self-administer stimulantsintravenously and that dopamine is critical in their reinforcingeffects. Recently NO containing neurons have been shown to co-localizewith dopamine in areas of the striatum and ventral tegmental area andthat NO can modulate stimulant-evoked dopamine (DA) release.Administration of dopamine D1 receptor antagonists decrease the levelsof striatal NADPH-diaphorase staining, a marker for NOS activity, whileD2 antagonists produce the opposite effect. L-Arginine, the substrate ofNOS, is also a potent modulator of DA release. Also, multipleNO-generating agents increase DA efflux or inhibit reuptake both invitro and in vivo. L-NAME has been shown to significantly alter cocainereinforcement by decreasing the amount of self-administration and byincreasing the inter-response time between successive cocaine injections(Pudiak and Bozarth, Soc. Neurosci. Abs. 22:703, 1996). This indicatesthat NOS inhibition by compounds of the invention may be useful in thetreatment of cocaine addiction.

(b) Morphine/Opioid Induced Tolerance and Withdrawal Symptoms

There is much evidence supporting the role of both the NMDA and NOpathways in opioid dependence in adult and infant animals. Adult orneonatal rodents injected with morphine sulfate develop behavioralwithdrawal after precipitation with naltrexone. The withdrawal symptomsafter naltrexone initiation can be reduced by administration of NOSinhibitors, such as 7-NI or L-NAME (Zhu and Barr, Psychopharmacology150(3):325-336, 2000). In a related study, it was shown that the morenNOS selective inhibitor 7-NI attenuated more of the morphine inducedwithdrawal symptoms including mastication, salivation and genitaleffects than the less selective compounds (Vaupel et al.,Psychopharmacology (Berl.) 118(4):361-8, 1995). This indicates that NOSinhibition by compounds of the invention may be useful in the treatmentof morphine/opioid induced tolerance and withdrawal symptoms.

(c) Ethanol Tolerance and Dependence

Among the factors that influence alcohol dependence, tolerance to theeffects of ethanol is an important component because it favors theexaggerated drinking of alcoholic beverages (Lê and Kiianmaa,Psychopharmacology (Berl.) 94:479-483, 1988). In a study with rats,ethanol tolerance to motor incoordination and hypothermia developrapidly and can be blocked by i.c.v. administration of 7-NI withoutaltering cerebral ethanol concentrations (Wazlawik and Morato, BrainRes. Bull. 57(2):165-70, 2002). In other studies, NOS inhibition withL-NAME (Rezvani et al., Pharmacol. Biochem. Behav. 50:265-270, 1995) orby i.c.v. injection of nNOS antisense (Naassila et. al., Pharmacol.Biochem. Behav. 67:629-36, 2000) reduced ethanol consumption in theseanimals. This indicates that NOS inhibition by compounds of theinvention may be useful in the treatment of ethanol tolerance anddependence.

Administration of a compound of the invention, either alone or incombination with another therapeutic agent, such as, for example, anNMDA antagonist, may be useful for the treatment of chemicaldependencies and drug addictions.

Epilepsy

Co-administration of 7-NI with certain anticonvulsants, such ascarbamazepine, shows a synergistic protective effect againstamygdala-kindled seizures in rats at concentrations that do not alterroto-rod performance (Borowicz et al., Epilepsia 41(9:112-8, 2000).Thus, an NOS inhibitor, such as, for example, a compound of theinvention, either alone or in combination with another therapeuticagent, such as, for example, an antiepileptic agent, may be useful forthe treatment of epilepsy or a similar disorder. Examples ofantiepileptic agents useful in a combination of the invention includecarbamazepine, gabapentin, lamotrigine, oxcarbazepine, phenyloin,topiramate, and valproate.

Diabetic Nephropathy

Urinary excretion of NO byproducts is increased in diabetic rats afterstreptozotocin treatment and increased NO synthesis has been suggestedto be involved in diabetic glomerular hyperfiltration. The neuronalisoform nNOS is expressed in the loop of Henle and mucula densa of thekidney and inhibition of this isoform using 7-NI reduces glomerularfiltration without affecting renal arteriole pressure or renal bloodflow (Sigmon et al., Gen. Pharmacol. 34(2):95-100, 2000). Both thenon-selective NOS inhibitor L-NAME and the nNOS selective 7-NI normalizerenal hyperfiltration in diabetic animals (Ito et al., J. Lab Clin. Med.138(3):177-185, 2001). Therefore, administration of a compound of theinvention may be useful for the treatment of diabetic nephropathy.

Medication Overuse Headache

Medication overuse headache (MOH) is associated with excessive use ofcombination analgesics, opioids, barbiturates, aspirin, NSAIDS, caffeineand triptans and is a common problem that limits the usefulness of thesetypes of medications (Diener and Limmroth. Medication-overuse headache:a worldwide problem. Lancet Neurol. 2004: 3, 475-483). It is generallydefined as headaches that present >15 days per month (HeadacheClassification Committee. The International Classification of HeadacheDisorders (2^(nd) Ed). Cephalalgia 2004: 24 (Supple. 1); 9-160). It iswell documented that acute treatment of patients for migraine or tensiontype headache are at increased risk of headache aggravation, developdaily headache, or may become refractory to treatment if the acutemedication is taken excessively (Zeeberg et. al. Cephalalgia 2006: 26,1192-1198). MOH patients generally are unresponsive to prophylacticmedications while overusing medications. Currently the treatment ofchoice for MOH is discontinuation of medication although this oftenassociated with withdrawal symptoms such as nausea, vomiting and sleepdisturbances. While migraine or tension-type headache patients sufferingfrom MOH that discontinue medication for 2 months have a reduction inheadache frequency (45%), many patients were either unchanged (48%)following withdrawal or had an aggravation of headache (Zeeberg et. al.Cephalalgia 2006: 26, 1192-1198). Thus there remains a large unmet needfor patients suffering from MOH.

It is believed that certain features of MOH, such as increased headachefrequency, expansion of headache area and the development of cutaneousallodynia are a result of medication-induced central sensitization oftrigeminal nociceptive pathways and periacqueductal grey area (Waeberand Moskowitz. Therapeutic implications of central and peripheralneurologic mechanisms in migraine. Neurology: 2003, 61(Suppl. 4);S9-20). Similar to behavioral sensitization to psychostimulants, therepeated administration of headache medications (e.g., triptans) resultsin cross-sensitization among different drugs used to treat headache.Changes in synaptic plasticity involve changes in intracellular calciumand nitric oxide levels. Patients suffering from chronic headache,migraine and MOH patients show increased levels of platelet nitratelevels. Thus the development of the sensitization in MOH is likelymediated by the changes in NO and calcium levels in the CNS (Sarchielliet. al. Nitric oxide pathway, Ca2+, and serotonin content in plateletsfrom patients suffering from chronic daily headache. Cephalalgia 1999:19; 810-816). Given that the development of central sensitization ismediated by nNOS (Cizkova et. al. Brain. Res. Bull. 2002; 58(2):161-171, Choi et. al. J. Neurol. Sci. 1996; 138(1-2): 14-20, as such, itis expected that neuronal nitric oxide synthase inhibitors, such as thecompounds of the invention, will be useful in the prevention andtreatment of MOH if used in concomitantly with other headachemedications. It is also expected that both CTTH and migraine treatmentwith nNOS inhibitors will not result in the development of MOH.

Gastrointestinal Disorders

nNOS constitutes more than 90% of the total NOS in the small intestine.Although iNOS is constitutively present, it accounts for less than 10%of the total NOS activity, and eNOS is essentially undetectable in theintestine (Qu X W et. al. Type I nitric oxide synthase (NOS) is thepredominant NOS in rat small intestine. Regulation byplatelet-activating factor. Biochim Biophys Acta 1999; 1451: 211-217).The main function of nNOS in the intestine is believed to be regulationof gut motility via neuronal signal transmission in the NANC componentsof the nervous system. NO regulates the muscle tone of the sphincter inthe lower esophagus, pylorus, sphincter of Oddi, and anus. NO alsoregulates the accommodation reflex of the fundus and the peristalticreflex of the intestine. NOS inhibitors are known to delay gastricemptying and colonic transit (T. Takahashi J. Gastroenterol. 2003;38(5):421-30). Thus nNOS inhibitors can be therapeutic in GI disordersthat would benefit from the delay of gastric emptying or slowing ofcolonic transit. Dumping syndrome is a disorder that in which food isemptied too quickly from the stomach, filling the small intestine withundigested food that is not adequately prepared to permit efficientabsorption of nutrients in the small intestine and is often observedafter gastrectomy. Therefore, administration of a compound of theinvention may be useful for the treatment of gastrointestinal disorderssuch as dumping syndrome. The compounds of the invention may also beemployed to treat other irritiable bowel syndromes.

Visceral Pain

Visceral pain is the most common form of pain and is one of the mostdifficult forms of pain to treat. Visceral pain is distinct from somaticpain and is generally described as pain that originates from the body'sinternal cavities or organs and has five important clinical and sensorycharacteristics: (1) it is not evoked from all visceral organs (e.g.,liver, kidney, lung); (2) it is not always linked to visceral injury(e.g., cutting an intestine does not evoke pain); (3) it is diffuse; (4)it is referred to other locations; and (5) it can be referred to otherautonomic and motor reflexes (e.g., nausea, lower-back muscle tensionfrom renal colic) (Lancet 353, 2145-48, 1999). Several theories havebeen proposed for the mechanisms of visceral pain. In the first theory,the viscera are innervated by separate classes of neurons, one concernedwith autonomic regulation and the other with sensory phenomena such aspain. The second theory suggests a single homogenous class of sensoryreceptors that are active at low frequencies (normal regulatory signals)or at high frequencies of activation (induced by intense pain signals).However, studies indicate that the viscera is innervated by two classesof nociceptive sensory receptors: high threshold (mostly mechanicalreceptors found in heart, vein, lung, airways, esophagus, bilary system,small intestine, colon, ureter, urinary bladder, and uterus, which areactivated by noxious stimuli) and low threshold intensity codingreceptors that respond to innocuous and noxious stimuli (heart,esophagus, colon, urinary bladder, and testes). Yet another theorysuggests a component of afferent fibres that are normally unresponsiveto stimuli (silent nociceptors) that can become activated or sensitizedduring inflammation (Trends Neurosci. 15, 374-78, 1992). Oncesensitized, these nociceptors now respond to innocuous stimuli thatnormally occur in the internal organs resulting in an enhanced barrageof convergent input to the spinal cord and subsequently triggeringcentral mechanisms that amplify the effect of the peripheral input.

Visceral pain can result from neoplasm, infection, or injury. Forexample, visceral pain may be caused by disease or injury to an internalorgan, which refers pain to other parts of the body. The compounds ofthe invention can be also used to treat visceral pain that is, forexample, secondary to irritable bowel syndrome, inflammatory bowelsyndrome, pancreatitis, diverticulitis, Crohn's disease, peritonitis,pericarditis, hepatitis, appendicitis, colitis, cholecystitis,gastroenteritis, endometriosis, dysmenorrhea, interstitial cystitis,prostatitis, pleuritis, upper gastrointestinal dyspepsia, renal colic,or biliary colic; visceral pain that is secondary to a disease of theliver, kidney, ovary, uterus, bladder, bowel, stomach, esophagus,duodenum, intestine, colon, spleen, pancreas, appendix, heart, orperitoneum; visceral pain that results from a neoplasm or injury, orvisceral pain that results from infection. Visceral pain treated by themethods of the invention may be inflammatory or non-inflammatory.

Visceral pain models and assays are known in the art (e.g., Bourdu etal., Gastroenterology 128:1996-2008, 2005; Vera-Portocarrero et al.,Gastroenterology 130:2155-2164, 2006; and Sparmann et al.,Gastroenterology 112:1664-1672, 1997).

Combination Formulations and Uses Thereof

In addition to the formulations described above, one or more compoundsof the invention can be used in combination with other therapeuticagents. For example, one or more compounds of the invention can becombined with another NOS inhibitor. Exemplary inhibitors useful forthis purpose include, without limitation, those described in U.S. Pat.Nos. 6,235,747, 7,141,595, and 7,375,219; U.S. patent application Ser.Nos. 09/127,158, 09/325,480, 09/403,177, 09/802,086, 09/826,132,09/740,385, 09/381,887, 10/476,958, 10/483,140, 10/484,960, 10/678,369,10/819,853, 10/938,891, 11/436,393, 11/787,167, 12/054,083, and12/272,656; International Publication Nos. WO 97/36871, WO 98/24766, WO98/34919, WO 99/10339, WO 99/11620, and WO 99/62883.

In another example, one or more compounds of the invention can becombined with an antiarrhythmic agent. Exemplary antiarrhythmic agentsinclude, without limitation, lidocaine and mixiletine.

GABA-B agonists, alpha-2-adrenergic receptor agonists, cholecystokininantagonists, 5HT_(1B/1D) agonists, or CGRP antagonists can also be usedin combination with one or more compounds of the invention. Non-limitingexamples of alpha-2-adrenergic receptor agonists include clonidine,lofexidine, and propanolol. Non-limiting examples of cholecystokininantagonists include L-365, 260; CI-988; LY262691; S0509, or thosedescribed in U.S. Pat. No. 5,618,811. Non-limiting examples of5HT_(1B/1D) agonists that may be used in combination with a compound ofthe invention include dihydroegotamine, eletriptan, frovatriptan,naratriptan, rizatriptan, sumatriptan, donitriptan, or zolmitriptan.Non-limiting examples of CGRP antagonists that may be used incombination with a compound of the invention include quinine analoguesas described in International Publication No. WO9709046, non-peptideantagonists as described in International Publication Nos. WO0132648,WO0132649, WO9811128, WO9809630, WO9856779, WO0018764, or otherantagonists such as SB-(+)-273779 or BIBN-4096BS.

Substance P antagonists, also known as NK₁ receptor antagonists, arealso useful in combination with one or more compounds of the invention.Exemplary inhibitors useful for this purpose include, withoutlimitation, those compounds disclosed in U.S. Pat. Nos. 3,862,114,3,912,711, 4,472,305, 4,481,139, 4,680,283, 4,839,465, 5,102,667,5,162,339, 5,164,372, 5,166,136, 5,232,929, 5,242,944, 5,300,648,5,310,743, 5,338,845, 5,340,822, 5,378,803, 5,410,019, 5,411,971,5,420,297, 5,422,354, 5,446,052, 5,451,586, 5,525,712, 5,527,811,5,536,737, 5,541,195, 5,594,022, 5,561,113, 5,576,317, 5,604,247,5,624,950, and 5,635,510; International Publication Nos. WO 90/05525, WO91/09844, WO 91/12266, WO 92/06079, WO 92/12151, WO 92/15585, WO92/20661, WO 92/20676, WO 92/21677, WO 92/22569, WO 93/00330, WO93/00331, WO 93/01159, WO 93/01160, WO 93/01165, WO 93/01169, WO93/01170, WO 93/06099, WO 93/10073, WO 93/14084, WO 93/19064, WO93/21155, WO 94/04496, WO 94/08997, WO 94/29309, WO 95/11895, WO95/14017, WO 97/19942, WO 97/24356, WO 97/38692, WO 98/02158, and WO98/07694; European Patent Publication Nos. 284942, 327009, 333174,336230, 360390, 394989, 428434, 429366, 443132, 446706, 484719, 499313,512901, 512902, 514273, 514275, 515240, 520555, 522808, 528495, 532456,and 591040.

Suitable classes of antidepressant agents that may be used incombination with a compound of the invention include, withoutlimitation, norepinephrine re-uptake inhibitors, selective serotoninre-uptake inhibitors (SSRIs), selective noradrenaline/norepinephrinereuptake inhibitors (NARIs), monoamine oxidase inhibitors (MAOs),reversible inhibitors of monoamine oxidase (RIMAs), dualserotonin/noradrenaline re-uptake inhibitors (SNRIs), α-adrenoreceptorantagonists, noradrenergic and specific serotonergic antidepressants(NaSSAs), and atypical antidepressants.

Non-limiting examples of norepinephrine re-uptake inhibitors includetertiary amine tricyclics and secondary amine tricyclics, such as, forexample, adinazolam, amineptine, amoxapine, butriptyline, demexiptiline,desmethylamitriptyline, desmethylclomipramine, demexiptiline,desipramine, doxepin, dothiepin, fluacizine, imipramine, imipramineoxide, iprindole, lofepramine, maprotiline, melitracen, metapramine,norclolipramine, nortriptyline, noxiptilin, opipramol, perlapine,pizotifen, pizotyline, propizepine, protriptyline, quinupramine,tianeptine, trimipramine, trimipramineamiltriptylinoxide, andpharmaceutically acceptable salts thereof.

Non-limiting examples of selective serotonin re-uptake inhibitorsinclude, for example, clomipramine, femoxetine, fluoxetine, fluvoxamine,paroxetine, and sertraline, and pharmaceutically acceptable saltsthereof.

Non-limiting examples of selective noradrenaline/norepinephrine reuptakeinhibitors include, for example, atomoxetine, bupropion; reboxetine,tomoxetine, and viloxazine and pharmaceutically acceptable saltsthereof.

Non-limiting examples of selective monoamine oxidase inhibitors include,for example, isocarboxazid, phenelzine, tranylcypromine and selegiline,and pharmaceutically acceptable salts thereof. Other monoamine oxidaseinhibitors useful in a combination of the invention include clorgyline,cimoxatone, befloxatone, brofaromine, bazinaprine, BW-616U (BurroughsWellcome), BW-1370U87 (Burroughs Wellcome), CS-722 (RS-722) (Sankyo),E-2011 (Eisai), harmine, harmaline, moclobemide, PharmaProjects 3975(Hoechst), RO 41-1049 (Roche), RS-8359 (Sankyo), T-794 (Tanabe Seiyaku),toloxatone, K-Y 1349 (Kalir and Youdim), LY-51641 (Lilly), LY-121768(Lilly), M&B 9303 (May & Baker), MDL 72394 (Marion Merrell), MDL 72392(Marion Merrell), sercloremine, and MO 1671, and pharmaceuticallyacceptable salts thereof. Suitable reversible inhibitors of monoamineoxidase that may be used in the present invention include, for example,moclobemide, and pharmaceutically acceptable salts thereof.

Non-limiting examples of dual serotonin/norepinephrine reuptake blockersinclude, for example, duloxetine, milnacipran, mirtazapine, nefazodone,and venlafaxine.

Non-limiting examples of other antidepressants that may be used in amethod of the present invention include adinazolam, alaproclate,amineptine, amitriptyline amitriptyline/chlordiazepoxide combination,atipamezole, azamianserin, bazinaprine, befuraline, bifemelane,binodaline, bipenamol, brofaromine, caroxazone, cericlamine,cianopramine, cimoxatone, citalopram, clemeprol, clovoxamine, dazepinil,deanol, demexiptiline, dibenzepin, dimetacrine, dothiepin, droxidopa,enefexine, estazolam, etoperidone, fengabine, fezolamine, fluotracen,idazoxan, indalpine, indeloxazine, levoprotiline, litoxetine;medifoxamine, metralindole, mianserin, minaprine, montirelin,nebracetam, nefopam, nialamide, nomifensine, norfluoxetine, orotirelin,oxaflozane, pinazepam, pirlindone, ritanserin, rolipram, sercloremine,setiptiline, sibutramine, sulbutiamine, sulpiride, teniloxazine,thozalinone, thymoliberin, tiflucarbine, tofenacin, tofisopam,toloxatone, veralipride, viqualine, zimelidine, and zometrapine, andpharmaceutically acceptable salts thereof, and St. John's wort herb, orHypencuin perforatum, or extracts thereof.

In another example, opioids can be used in combination with one or morecompounds of the invention. Exemplary opioids useful for this purposeinclude, without limitation, alfentanil, butorphanol, buprenorphine,dextromoramide, dezocine, dextropropoxyphene, codeine, dihydrocodeine,diphenoxylate, etorphine, fentanyl, hydrocodone, hydromorphone,ketobemidone, loperamide, levorphanol, levomethadone, meperidine,meptazinol, methadone, morphine, morphine-6-glucuronide, nalbuphine,naloxone, oxycodone, oxymorphone, pentazocine, pethidine, piritramide,propoxylphene, remifentanil, sulfentanyl, tilidine, and tramadol.

In yet another example, anti-inflammatory compounds, such as steroidalagents or non-steroidal anti-inflammatory drugs (NSAIDs), can be used incombination with one or more compounds of the invention. Non-limitingexamples of steroidal agents include prednisolone and cortisone.Non-limiting examples of NSAIDs include acemetacin, aspirin, celecoxib,deracoxib, diclofenac, diflunisal, ethenzamide, etofenamate, etoricoxib,fenoprofen, flufenamic acid, flurbiprofen, lonazolac, lornoxicam,ibuprofen, indomethacin, isoxicam, kebuzone, ketoprofen, ketorolac,naproxen, nabumetone, niflumic acid, sulindac, tolmetin, piroxicam,meclofenamic acid, mefenamic acid, meloxicam, metamizol, mofebutazone,oxyphenbutazone, parecoxib, phenidine, phenylbutazone, piroxicam,propacetamol, propyphenazone, rofecoxib, salicylamide, suprofen,tiaprofenic acid, tenoxicam, valdecoxib,4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide,N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide,2-(3,4-difluorophenyl)-4-(3-hydroxy-3-methylbutoxy)-5-[4-(methylsulfonyl)phenyl]-3(2H)-pyridazinone,and2-(3,5-difluorophenyl)-3-[4-(methylsulfonyl)phenyl]-2-cyclopenten-1-one).Compounds of the invention may also be use in combination withacetaminophen.

Any of the above combinations can be used to treat any appropriatedisease, disorder, or condition. Exemplary uses for combinations of acompound of the invention and another therapeutic agent are describedbelow.

Opioid-NOS Inhibitor Combinations in Chronic, Neuropathic Pain

Nerve injury can lead to abnormal pain states known as neuropathic pain.Some of the clinical symptoms include tactile allodynia (nociceptiveresponses to normally innocuous mechanical stimuli), hyperalgesia(augmented pain intensity in response to normally painful stimuli), andspontaneous pain. Spinal nerve ligation (SNL) in rats is an animal modelof neuropathic pain that produces spontaneous pain, allodynia, andhyperalgesia, analogous to the clinical symptoms observed in humanpatients (Kim and Chung, Pain 50:355-363, 1992; Seltzer, Neurosciences7:211-219, 1995).

Neuropathic pain can be particularly insensitive to opioid treatment(Benedetti et al., Pain 74:205-211, 1998) and is still considered to berelatively refractory to opioid analgesics (MacFarlane et al.,Pharmacol. Ther. 75:1-19, 1997; Watson, Clin. J. Pain 16:S49-S55, 2000).While dose escalation can overcome reduced opioid effectiveness, it islimited by increased side effects and tolerance. Morphine administrationis known to activate the NOS system, which limits the analgesic actionof this drug (Machelska et al., NeuroReport 8:2743-2747, 1997; Wong etal., Br. J. Anaesth. 85:587, 2000; Xiangqi and Clark, Mol. Brain. Res.95:96-102, 2001). However, it has been shown that the combined systemicadministration of morphine and L-NAME can attenuate mechanical and coldallodynia at subthreshold doses at which neither drug administered alonewas effective (Ulugol et al., Neurosci. Res. Com. 30(3):143-153, 2002).The effect of L-NAME co-administration on morphine analgesia appears tobe mediated by nNOS, as L-NAME loses its ability to potentiate morphineanalgesia in nNOS null-mutant mice (Clark and Xiangqi, Mol. Brain. Res.95:96-102, 2001). Enhanced analgesia has been demonstrated in thetail-flick or paw pressure models using coadministration of L-NAME or7-NI with either a mu-, delta-, or kappa-selective opioid agonist(Machelska et al., J. Pharmacol. Exp. Ther. 282:977-984, 1997).

While opioids are an important therapy for the treatment of moderate tosevere pain, in addition to the usual side effects that limit theirutility, the somewhat paradoxical appearance of opioid-inducedhyperalgesia may actually render patients more sensitive to pain andpotentially aggravate their pain (Angst and Clark, Anesthesiology, 2006,104(3), 570-587; Chu et. al. J. Pain 2006, 7(1) 43-48). The developmentof tolerance and opioid induced hyperalgesia is consistent withincreased levels of NO production in the brain. The reduced analgesicresponse to opioids is due to an NO-induced upregulated hyperalgesicresponse (Heinzen and Pollack, Brain Res. 2004, 1023, 175-184).

Thus, the combination of an nNOS inhibitor with an opioid (for example,those combinations described above) can enhance opioid analgesia inneuropathic pain and prevent the development of opioid tolerance andopioid-induced hyperalgesia.

Antidepressant-NOS Inhibitor Combinations for Chronic Pain, NeuropathicPain, Chronic Headache or Migraine

Many antidepressants are used for the treatment of neuropathic pain(McQuay et al., Pain 68:217-227, 1996) and migraine (Tomkins et al., Am.J. Med. 111:54-63, 2001), and act via the serotonergic or noradrenergicsystem. NO serves as a neuromodulator of these systems (Garthwaite andBoulton, Annu. Rev. Physiol. 57:683, 1995). 7-NI has been shown topotentiate the release of noradrenaline (NA) by the nicotinicacetylcholine receptor agonist DMPP via the NA transporter (Kiss et al.,Neuroscience Lett. 215:115-118, 1996). It has been shown that localadministration of antidepressants, such as paroxetine, tianeptine, andimipramine decrease levels of hippocampal NO (Wegener et al., Brain Res.959:128-134, 2003). It is likely that NO is important in the mechanismby which antidepressants are effective for treating pain and depression,and that a combination of an nNOS inhibitor with an antidepressant, suchas, for example, those combinations described above, will produce bettertreatments.

Serotonin 5HT_(1B/1D/1F) Agonist or CGRP Antagonist and NOS InhibitorCombinations in Migraine

Administration of glyceryl trinitrate (GTN), an NO donor, inducesimmediate headaches in normal individuals and results in delayedmigraine attacks in migraineurs with a 4-6 hour latency period (Iversenet al., Pain 38:17-24, 1989). In patients with migraine attack, levelsof CGRP (Calcitonin Gene Related Peptide), a potent vasodialator, in thecarotid artery correlate with the onset and ablation of migraine attack(Durham, Curr Opin Investig Drugs 5(7):731-5, 2004). Sumatriptan, anantimigraine drug having affinity at 5HT_(1B), 5HT_(1D), and 5HT_(1F)receptors, reduces GTN-induced immediate headache and in parallelcontracts cerebral and extracerebral arteries (Iversen and Olesen,Cephalagia 13(Suppl 13):186, 1993). The antimigraine drug rizatriptanalso reduces plasma levels of CGRP following migraine pain reduction(Stepien et al., Neurol. Neurochir. Pol. 37(5):1013-23, 2003). Both NOand CGRP have therefore been implicated as a cause for migraine.Serotonin 5HT_(1B/1D) agonists have been shown to block NMDAreceptor-evoked NO signaling in brain cortex slices (Strosznajder etal., Cephalalgia 19(10):859, 1999). These results suggest that acombination of a compound of the invention and a selective ornon-selective 5HT_(1B/1D/1F) agonist or a CGRP antagonist, such as thosecombinations described above, would be useful for the treatment ofmigraine.

Pharmaceutical Compositions

The compounds of the invention are preferably formulated intopharmaceutical compositions for administration to human subjects in abiologically compatible form suitable for administration in vivo.Accordingly, in another aspect, the present invention provides apharmaceutical composition comprising a compound of the invention inadmixture with a suitable diluent, carrier, or excipient.

The compounds of the invention may be used in the form of the free base,in the form of salts, solvates, and as prodrugs. All forms are withinthe scope of the invention. In accordance with the methods of theinvention, the described compounds or salts, solvates, or prodrugsthereof may be administered to a patient in a variety of forms dependingon the selected route of administration, as will be understood by thoseskilled in the art. The compounds of the invention may be administered,for example, by oral, parenteral, buccal, sublingual, nasal, rectal,patch, pump, or transdermal administration and the pharmaceuticalcompositions formulated accordingly. Parenteral administration includesintravenous, intraperitoneal, subcutaneous, intramuscular,transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topicalmodes of administration. Parenteral administration may be by continuousinfusion over a selected period of time.

A compound of the invention may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or it maybe enclosed in hard or soft shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, a compound of theinvention may be incorporated with an excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

A compound of the invention may also be administered parenterally.Solutions of a compound of the invention can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO and mixtures thereof with or without alcohol, and in oils.Under ordinary conditions of storage and use, these preparations maycontain a preservative to prevent the growth of microorganisms.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003—20th edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19),published in 1999.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that may be easily administered via syringe.

Compositions for nasal administration may conveniently be formulated asaerosols, drops, gels, and powders. Aerosol formulations typicallyinclude a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device, such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant, which can be a compressed gas,such as compressed air or an organic propellant, such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, where the active ingredient isformulated with a carrier, such as sugar, acacia, tragacanth, or gelatinand glycerine. Compositions for rectal administration are convenientlyin the form of suppositories containing a conventional suppository base,such as cocoa butter.

The compounds of the invention may be administered to an animal, e.g., ahuman, alone or in combination with pharmaceutically acceptablecarriers, as noted above, the proportion of which is determined by thesolubility and chemical nature of the compound, chosen route ofadministration, and standard pharmaceutical practice.

The dosage of the compounds of the invention, and/or compositionscomprising a compound of the invention, can vary depending on manyfactors, such as the pharmacodynamic properties of the compound; themode of administration; the age, health, and weight of the recipient;the nature and extent of the symptoms; the frequency of the treatment,and the type of concurrent treatment, if any; and the clearance rate ofthe compound in the animal to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors. Thecompounds of the invention may be administered initially in a suitabledosage that may be adjusted as required, depending on the clinicalresponse. In general, satisfactory results may be obtained when thecompounds of the invention are administered to a human at a daily dosageof between 0.05 mg and 3000 mg (measured as the solid form). A preferreddose ranges between 0.05-500 mg/kg, more preferably between 0.5-50mg/kg.

A compound of the invention can be used alone or in combination withother agents that have NOS-inhibiting activity, or in combination withother types of treatment (which may or may not inhibit NOS) to treat,prevent, and/or reduce the risk of stroke, neuropathic or migraine pain,or other disorders that benefit from NOS inhibition. In combinationtreatments, the dosages of one or more of the therapeutic compounds maybe reduced from standard dosages when administered alone. In this case,dosages of the compounds when combined should provide a therapeuticeffect.

In addition to the above-mentioned therapeutic uses, a compound of theinvention can also be used in diagnostic assays, screening assays, andas a research tool.

In diagnostic assays, a compound of the invention may be useful inidentifying or detecting NOS activity. For such a use, the compound maybe radiolabeled (as described elsewhere herein) and contacted with apopulation of cells of an organism. The presence of the radiolabel onthe cells may indicate NOS activity.

In screening assays, a compound of the invention may be used to identifyother compounds that inhibit NOS, for example, as first generationdrugs. As research tools, the compounds of the invention may be used inenzyme assays and assays to study the localization of NOS activity. Suchinformation may be useful, for example, for diagnosing or monitoringdisease states or progression. In such assays, a compound of theinvention may also be radiolabeled.

NOS In Vitro Inhibition Assays

The compounds of the present invention have been found to exhibitselective inhibition of the neuronal isoform of NOS (nNOS). Compoundsmay be examined for their efficacy in preferentially inhibiting nNOSover iNOS and/or eNOS by a person skilled in the art, for example, byusing the methods described in Example 34 and herein below.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Synthesis ofN-(4-(2-(1-methylpyrrolidin-2-yl)ethyl)-3-oxochroman-7-yl)thiophene-2-carboximidamide(1)

7-Nitro-2H-benzo[b][1,4]oxazin-3(4H)-one

Prepared according to reported procedure in J. Chem. Research (M) 2003,1120-1128.

4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one

A suspension of 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (0.5 g, 2.575mmol), 2-(2-chloroethyl)-1-methylpyrrolidine hydrochloride (0.94 g,5.150 mmol), NaI (0.19 g, 1.287 mmol), and K₂CO₃ (2.13 g, 15.452 mmol)in dry DMF (10 mL) was stirred at room temperature overnight (18 hours).The reaction was diluted with water (150 mL), and the product wasextracted into ethyl acetate (2×25 mL). The combined ethyl acetatelayers were washed with brine (20 mL) and dried (Na₂SO₄). The solventwas evaporated, and the crude material was purified by columnchromatography (3:97 (2M NH₃ in MeOH):CH₂Cl₂) to obtain the titlecompound (0.525 g, 67%) as a yellow solid. ¹H NMR (DMSO-d₆) δ 1.47-1.65(m, 4H), 1.79-2.13 (m, 4H), 2.18 (s, 3H), 2.89-2.95 (m, 1H), 3.98 (t,2H, J=7.8 Hz), 4.79 (s, 2H), 7.40 (d, 1H, J=9.0 Hz), 7.80 (d, 1H, J=2.7Hz), 7.98 (dd, 1H, J=2.4, 9.0 Hz); ESI-MS (m/z, %): 306 (MH⁺, 100).

7-Amino-4-(2-(1-methylpyrrolidin-2-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one

A solution of4-(2-(1-methylpyrrolidin-2-yl)ethyl)-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one(0.49 g, 1.604 mmol) in dry ethanol (5 mL) was treated with Pd—C (˜0.05g) and purged with hydrogen gas. The flask was evacuated and purgedtwice with hydrogen gas. The reaction stirred at room temperature underhydrogen atmosphere (balloon pressure) for 2 hours. The reaction wasfiltered through a Celite bed and washed with methanol (3×10 mL). Thecombined organic layers were evaporated to obtain the crude titlecompound (0.44 g, quantitative) as a solid. ¹H NMR (DMSO-d₆) δ 1.39-1.51(m, 2H), 1.56-1.66 (m, 2H), 1.76-2.06 (m, 4H), 2.16 (s, 3H), 2.88-2.94(m, 1H), 3.74-3.86 (m, 2H), 4.46 (s, 2H), 5.01 (s, 2H), 6.22 (d, 1H,J=2.4 Hz), 6.26 (dd, 1H, J=2.1, 8.4 Hz), 6.82 (d, 1H, J=8.4 Hz); ESI-MS(m/z, %): 276 (MH⁺, 100).

N-(4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-3-oxochroman-7-yl)thiophene-2-carboximidamide(1)

A solution of7-amino-4-(2-(1-methylpyrrolidin-2-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one(0.12 g, 0.435 mmol) in dry ethanol (5 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.24 g, 0.871 mmol) at roomtemperature. The resulting mixture was stirred over night (18 hours). Atthis time, additional methyl thiophene-2-carbimidothioate hydroiodide(0.24 g, 0.871 mmol) was added, and the reaction stirred for anadditional 24 hours. The reaction was diluted with saturated NaHCO₃solution (25 mL), and the product was then extracted into CH₂Cl₂ (2×20mL). The combined CH₂Cl₂ layers was washed with brine (15 mL) and dried(Na₂SO₄). The solvent was evaporated, and the crude material waspurified by column chromatography (5:95 (2 M NH₃ in MeOH):CH₂Cl₂) toobtain title compound 1 (0.155 g, 93%) as a solid. ¹H NMR (DMSO-d₆) δ1.46-1.65 (m, 4H), 1.64-2.13 (m, 4H), 2.20 (s, 3H), 2.90-2.96 (m, 1H),3.86-3.93 (m, 2H), 4.58 (s, 2H), 6.49-6.58 (m, 4H), 7.07-7.10 (m, 2H),7.60 (d, 1H, J=5.4 Hz), 7.73 (d, 1H, J=3.0 Hz); ESI-MS (m/z, %): 385(MH⁺, 68), 274 (46), 193 (100); ESI-HRMS calculated for C₂₀H₂₅N₄O₂S(MH⁺), calculated: 385.1692, observed: 385.1687; HPLC purity 98% byarea.

Example 2 Synthesis ofN-(4-(2-(1-methylpyrrolidin-2-yl)ethyl)chroman-7-yl)thiophene-2-carboximidamide(2)

7-Amino-4-(2-(1-methylpyrrolidin-2-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one

For complete experimental details and spectral data, see Example 1.

4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-amine

A solution of LiAlH₄ (4.21 mL, 4.212 mmol, 1.0 M solution in THF) wastreated dropwise with7-amino-4-(2-(1-methylpyrrolidin-2-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one(0.29 g, 1.053 mmol) in dry THF (5 mL) at 0° C. The resulting mixturewas brought to room temperature and stirred overnight. The reaction wascarefully quenched with the sequential addition of water (0.16 mL), 2 NNaOH solution (0.16 mL), and water (0.16 mL). The reaction was filteredafter stirring for 30 minutes at room temperature and washed with CH₂Cl₂(3×10 mL). The combined CH₂Cl₂ layers were evaporated, and the crudematerial was purified by column chromatography (5:95 (2M NH₃ inMeOH):CH₂Cl₂) to obtain the title compound (0.23 g, 84%) as a syrup. ¹HNMR (DMSO-d₆) δ 1.28-1.45 (m, 2H), 1.56-1.65 (m, 2H), 1.75-2.10 (m, 4H),2.18 (s, 3H), 2.88-2.95 (m, 1H), 3.03-3.10 (m, 4H), 4.08 (t, 2H, J=4.8Hz), 4.36 (s, 2H), 6.00 (d, 1H, J=2.4 Hz), 6.06 (dd, 1H, J=2.4, 8.5 Hz),6.43 (d, 1H, J=8.4 Hz); ESI-MS (m/z, %): 262 (MH⁺, 100), 163 (33), 112(42).

N-(4-(2-(1-Methylpyrrolidin-2-yl)ethyl)chroman-7-yl)thiophene-2-carboximidamide(2)

A solution of4-(2-(1-methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-amine(0.21 g, 0.803 mmol) in dry ethanol (5 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.45 g, 1.606 mmol) at roomtemperature. The resulting mixture was stirred at room temperature for24 hours. The reaction was diluted with saturated NaHCO₃ solution (20mL), and the product was extracted into CH₂Cl₂ (2×20 mL). The combinedCH₂Cl₂ layers were washed with brine (15 mL) and dried (Na₂SO₄). Thesolvent was evaporated, and the crude material was purified by columnchromatography (5:95 (2 M NH₃ in MeOH):CH₂Cl₂) to obtain the titlecompound 2 (0.22 g, 75%) as a solid. ¹H NMR (DMSO-d₆) δ 1.35-1.52 (m,2H), 1.58-1.68 (m, 2H), 1.82-2.08 (m, 4H), 2.21 (s, 3H), 2.90-2.97 (m,1H), 3.18-3.32 (m, 4H), 4.16 (t, 2H, J=4.5 Hz), 6.22-6.34 (m, 4H), 6.63(d, 1H, J=8.4 Hz), 7.06 (dd, 1H, J=3.9, 5.1 Hz), 7.55 (d, 1H, J=5.1 Hz),7.68 (d, 1H, J=3.6 Hz); ESI-MS (m/z, %): 371 (MH⁺, 86), 260 (56), 186(100), 128 (81); ESI-HRMS calculated for C₂₀H₂₇N₄OS (MH⁺), calculated:371. 1900, observed: 371.1903; HPLC purity 98.2% by area.

Example 3 Synthesis ofN-(3-oxo-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(3)

7-Nitro-2H-benzo[b][1,4]oxazin-3(4H)-one

Prepared according to reported procedure in J. Chem. Research (M) 2003,1120-1128.

7-Nitro-4-(2-(pyrrolidin-1-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one

A suspension of 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (0.5 g, 2.575mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (0.43 g, 2.575 mmol)and K₂CO₃ (1.06 g, 7.726 mmol) in dry DMF (5 mL) was stirred at 100° C.overnight (18 hours). The reaction was brought to room temperature,diluted with water (100 mL), and the product was extracted into ethylacetate (2×25 mL). The combined ethyl acetate layers were washed withbrine (20 mL) and dried (Na₂SO₄). The solvent was evaporated, and thecrude material was purified by column chromatography (3:97 (2M NH₃ inMeOH):CH₂Cl₂) to obtain the title compound (0.49 g, 65%) as a solid. ¹HNMR (DMSO-d₆) δ 1.62-1.70 (m, 4H), 2.46-2.54 (m, 4H, merged with DMSO-d₆resonance), 2.60 (t, 2H, J=6.9 Hz), 4.08 (t, 2H, J=6.9 Hz), 4.80 (s,2H), 7.44 (d, 1H, J=9.3 Hz), 7.80 (d, 1H, J=2.4 Hz), 7.96 (dd, 1H,J=2.7, 9.0 Hz); ESI-MS (m/z, %): 292 (MH⁺, 100).

7-Amino-4-(2-(pyrrolidin-1-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one

A solution of7-nitro-4-(2-(pyrrolidin-1-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one(0.46 g, 1.579 mmol) in dry ethanol (5 mL) was treated with Pd—C (˜0.05g) and purged with hydrogen gas. The reaction was stirred under hydrogenatmosphere (balloon pressure) for 4 hours. The reaction was filteredthrough a Celite bed and washed with methanol (3×10 mL). The combinedorganic layers were evaporated and dried under vacuum to obtain thecrude title compound (0.4 g, 97%) as a solid. ¹H NMR (DMSO-d₆) δ1.61-1.70 (m, 4H), 2.44-2.56 (m, 6H, merged with DMSO-d₆), 3.90 (t, 2H,J=7.2 Hz), 4.47 (s, 2H), 5.01 (s, 2H), 6.22 (d, 1H, J=2.4 Hz), 6.25 (dd,1H, J=2.4, 8.2 Hz), 6.84 (d, 1H, J=8.7 Hz); ESI-MS (m/z, %): 262 (MH⁺,100), 191 (44).

N-(3-Oxo-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(3)

A solution of7-amino-4-(2-(pyrrolidin-1-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one(0.125 g, 0.478 mmol) in dry ethanol (5 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.27 g, 0.956 mmol) at roomtemperature. The resulting mixture was stirred for 2 days. The reactionwas then diluted with saturated NaHCO₃ solution (20 mL), and the productwas extracted into CH₂Cl₂ (2×20 mL). The combined CH₂Cl₂ layers werewashed with brine (15 mL) and dried (Na₂SO₄). The solvent wasevaporated, and the crude material was purified by column chromatography(3:97 (2 M NH₃ in MeOH):CH₂Cl₂) to obtain the title compound 3 (0.16 g,90%) as a solid. ¹H NMR (DMSO-d₆) δ 1.64-1.72 (m, 4H), 2.46-2.54 (m, 4H,merged with DMSO-d₆ resonance), 2.60 (t, 2H, J=7.2 Hz), 3.99 (t, 2H,J=7.2 Hz), 4.58 (s, 2H), 6.47-6.57 (m, 4H), 7.06-7.12 (m, 2H), 7.60 (d,1H, J=4.2 Hz), 7.73 (d, 1H, J=3.3 Hz); ESI-MS (m/z, %): 371 (MH⁺, 66),150 (63), 128 (100); ESI-HRMS calculated for C₁₉H₂₃N₄O₂S (MH⁺),calculated: 371.1536, observed: 371.1548; HPLC purity 93.17% by area.

Example 4 Synthesis ofN-(4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(4)

7-Nitro-2H-benzo[b][1,4]oxazin-3(4H)-one

Prepared according to a literature procedure in J. Chem. Research (M)2003, 1120-1128.

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine

A suspension of 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (300 mg, 1.545mmol) in THF (3 mL) was treated with BH₃-THF complex (15.45 mL, 15.45mmol, 1.0 M in THF), and the resulting orange solution was refluxedovernight. The reaction was cooled in an ice-bath. MeOH (15 mL) wasadded, and the reaction was then concentrated. A second portion of MeOH(20 mL) was added, and the solution was refluxed for 2 hours. At thistime, the reaction was concentrated, and the residue was subjected toflash chromatography on silica gel (10% EtOAc:90% hexane followed by 30%EtOAc:70% hexane). An orange solid was obtained after drying underreduced pressure. (240 mg, 86%). ¹H-NMR (DMSO-d₆) δ 7.68 (dd, J=2.4, 8.9Hz, 1H), 7.54 (s, 1H), 7.47 (d, J=2.4 Hz, 1H), 6.63 (d, J=9.0 Hz, 1H),4.15 (t, J=4.2 Hz, 2H), 3.45-3.40 (m, 2H).

7-Nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine

A solution of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine (300 mg, 1.665mmol) in DMF (10 mL) was treated with NaH (213 mg, 5.33 mmol, 60% wt inmineral oil) at 0° C., resulting in a bright orange suspension. Themixture was stirred for 10 minutes. 2-chloroethyl-pyrrolidinehydrochloride (566 mg, 3.33 mmol) was then added, and the reactionturned into a bright red suspension. The reaction was heated to 90° C.for 1 hour and then cooled to room temperature. The mixture was thendiluted with water (20 mL), transferred to a separatory funnel, andextracted into EtOAc (2×15 mL). The combined organic layers were washedwith brine (3×5 mL), dried (Na₂SO₄), filtered, and concentrated. Theresidue was subjected to flash chromatography on silica gel using CH₂Cl₂followed by 5:95 (2M NH₃ in MeOH):CH₂Cl₂ to give a solid (330 mg, 72%).¹H-NMR (DMSO-d₆) δ 7.75 (dd, J=2.7, 9.3 Hz, 1H), 7.47 (d, J=2.7 Hz, 1H),6.80 (d, J=9.0 Hz, 1H), 4.18 (t, J=4.2 Hz, 2H), 3.58-3.55 (m, 4H), 2.63(t, J=6.9 Hz, 2H), 2.58-2.48 (m, 4H), 1.70-1.63 (m, 4H); ESI-MS (m/z,%): 278 (MH⁺, 100), 98 (20).

N-(4-(2-(Pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(4)

A suspension of7-nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine(330 mg, 1.190 mmol) and Pd—C (252 mg, 0.238 mmol, 10% wt) in dry EtOH(10 mL) was purged with hydrogen gas. The reaction was stirred at roomtemperature for 1.5 hours under a hydrogen atmosphere (balloonpressure). The reaction mixture was filtered through a Celite pad andwashed with EtOH (25 mL). The filtrate was treated with methylthiophene-2-carbimidothioate hydroiodide (0.678 g, 2.377 mmol) andstirred for three days at room temperature. The reaction was dilutedwith saturated NaHCO₃ solution (50 mL), and the product was extractedinto CH₂Cl₂ (3×25 mL). The combined organic layers were dried (Na₂SO₄)and concentrated. The residue was subjected to flash chromatography onsilica gel using 2:98 MeOH:CH₂Cl₂ followed by 5:95 (2M NH₃ inMeOH):CH₂Cl₂ to give a yellow solid (150 mg, 36%). ¹H-NMR (DMSO-d₆) δ7.70 (d, J=3.6 Hz, 1H), 7.57 (dd, J=5.1, 0.9 Hz, 1H), 7.08 (dd, J=5.1,3.9 Hz, 1H), 6.65 (d, J=8.4 Hz, 1H), 6.34 (dd, J=2.4, 8.4 Hz, 1H), 6.252(d, J=2.4, 1H), 6.36-6.25 (m, 2H), 4.15 (t, J=4.2 Hz, 2H), 3.38-3.30 (m,4H), 2.64 (t, J=7.2 Hz, 2H), 2.58-2.48 (m, 4H), 1.70-1.67 (m, 4H);ESI-MS (m/z, %): 357 (MH⁺, 100), 260 (40), 98 (27); ESI-HRMS calculatedfor C₁₉H₂₅N₄OS (MH⁺): 357.1743, observed: 357.1734; HPLC purity: 95.8%by area.

Example 5N-(4-(2-(Dimethylamino)ethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(5)

7-Nitro-2H-benzo[b][1,4]oxazin-3(4H)-one

Prepared according to reported procedure in J. Chem. Research (M) 2003,1120-1128.

4-(2-(Dimethylamino)ethyl)-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one

A suspension of 7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (0.5 g, 2.575mmol), 2-chloro-N,N-dimethylethanamine hydrochloride (0.37 g, 2.575mmol), and K₂CO₃ (1.06 g, 7.726 mmol) in dry DMF (5 mL) was stirred at100° C. overnight (18 hours). The reaction was brought to roomtemperature, diluted with water (100 mL), and the product was extractedinto ethyl acetate (2×25 mL). The combined ethyl acetate layers werewashed with brine (20 mL) and then dried (Na₂SO₄). The solvent wasevaporated, and the crude material was purified by column chromatography(3:97 (2M NH₃ in MeOH):CH₂Cl₂) to obtain the title compound (0.52 g,76%) as a yellow solid. ¹H NMR (DMSO-d₆) δ 2.17 (s, 6H), 2.42 (t, 2H,J=6.6 Hz), 4.07 (t, 2H, J=6.6 Hz), 4.79 (s, 2H), 7.45 (d, 1H, J=9.0 Hz),7.80 (d, 1H, J=2.7 Hz), 7.96 (dd, 1H, J=2.4, 9.0 Hz); ESI-MS (m/z, %):266 (MH⁺, 100), 221 (66).

7-Amino-4-(2-(dimethylamino)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one

A solution of4-(2-(dimethylamino)ethyl)-7-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (0.5g, 1.884 mmol) in dry ethanol (5 mL) was treated with Pd—C (˜0.05 g) andpurged with hydrogen gas. The flask was evacuated and purged withhydrogen gas (twice) and stirred at room temperature under hydrogen atm.(balloon pressure) for 2 hours. The reaction was filtered through aCelite bed and washed with methanol (3×10 mL). The combined organiclayers were evaporated to obtain the crude title compound (0.44 g,quantitative) as a solid. ¹H NMR (DMSO-d₆) δ 2.16 (s, 6H), 2.36 (t, 2H,J=6.9 Hz), 3.88 (t, 2H, J=6.9 Hz), 4.47 (s, 2H), 5.01 (s, 2H), 6.21 (d,1H, J=2.1 Hz), 6.25 (dd, 1H, J=2.7, 8.5 Hz), 6.84 (d, 1H, J=8.4 Hz);ESI-MS (m/z, %): 236 (MH⁺, 31), 191 (100).

N-(4-(2-(Dimethylamino)ethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(5)

A solution of7-amino-4-(2-(dimethylamino)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one(0.125 g, 0.531 mmol) in dry ethanol (10 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.3 g, 1.062 mmol) at roomtemperature and stirred overnight (18 hours). At this time, additionalmethyl thiophene-2-carbimidothioate hydroiodide (0.3 g, 1.062 mmol) wasadded, and stirring was continued for another 24 hours. The reaction wasdiluted with saturated NaHCO₃ solution (20 mL), and the product wasextracted into CH₂Cl₂ (2×20 mL). The combined CH₂Cl₂ layers were washedwith brine (15 mL) and dried (Na₂SO₄). The solvent was evaporated, andthe crude material was purified by column chromatography (5:95 (2M NH₃in MeOH):CH₂Cl₂) to obtain the title compound 5 (0.16 g, 88%) as asolid. ¹H NMR (DMSO-d₆) δ 2.19 (s, 6H), 2.43 (t, 2H, J=6.9 Hz), 3.97 (t,2H, J=6.9 Hz), 4.58 (s, 2H), 6.48-6.58 (m, 4H), 7.06-7.14 (m, 2H), 7.60(d, 1H, J=5.1 Hz), 7.74 (d, 1H, J=3.3 Hz); ESI-MS (m/z, %): 345 (MH⁺,100), 128 (85); ESI-HRMS calculated for C₁₇H₂₁N₄O₂S (MH⁺), calculated:345.1379; observed: 345.1384; HPLC-purity: 93.15% by area.

Example 6 Synthesis ofN-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(6)

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine

For complete experimental details and spectral data, see example 4.

N,N-Dimethyl-2-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanamine

A solution of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine (240 mg, 1.33mmol) in DMF (10 mL) was treated with NaH (170 mg, 4.26 mmol, 60% wt inmineral oil) at 0° C., resulting in an orange mixture. The mixture wasthen treated with 2-chloro-N,N-dimethylethanamine hydrochloride (384 mg,2.66 mmol), resulting in a dark red mixture. The reaction was stirred atroom temperature for 1 hour. At this time, the reaction was then heatedto 90° C. and stirred for another 45 minutes. The reaction was cooled toroom temperature, water (15 mL) was added, and the reaction wasextracted into EtOAc (2×12 mL). The combined organic layers were washedwith brine (3×5 mL), dried (Na₂SO₄), filtered, and concentrated. Theresidue was subjected to flash chromatography on silica gel: CH₂Cl₂followed by 5:95 (2M NH₃ in MeOH):CH₂Cl₂ to give a yellow/orange solid(210 mg, 63%). ¹H-NMR (DMSO-d₆) δ 7.75 (dd, J=2.7, 9.0 Hz, 1H), 7.48 (d,J=2.7 Hz, 1H), 6.82 (d, J=9.0 Hz, 1H), 4.18 (t, J=4.5 Hz, 2H), 3.57-3.53(m, 4H), 2.54-2.45 (m, 2H), 2.23 (s, 6H).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(6)

A suspension ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanamine (210mg, 0.836 mmol) and Pd—C (10% wt, 89 mg, 0.084 mmol) in dry EtOH (15 mL)was purged with hydrogen gas. The reaction was stirred at roomtemperature for 3 hours under hydrogen atmosphere (balloon pressure).The reaction mixture was then filtered through a Celite pad and washedwith EtOH (15 mL). The light purple filtrate was concentrated to givecrude4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-amine. Asolution of this crude4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-amine(185 mg, 0.836 mmol) in dry EtOH (10 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (477 mg, 1.672 mmol) andstirred overnight at room temperature. The reaction was diluted withsaturated NaHCO₃ solution (50 mL) and the product was extracted intoCH₂Cl₂ (3×25 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated. The residue was subjected to flash chromatography onsilica gel using 2:98 MeOH:CH₂Cl₂, followed by 5:95 (2M NH₃ inMeOH):CH₂Cl₂, to give a yellow solid (150 mg, 54%). ¹H-NMR (DMSO-d₆) δ7.69 (dd, J=3.6, 0.9 Hz, 1H), 7.56 (dd, J=5.1, 0.9 Hz, 1′-1), 7.07 (dd,J=5.1, 3.9 Hz, 1H), 6.64 (d, J=8.4 Hz, 1H), 6.35-6.26 (m, 4H), 4.14 (t,J=3.9 Hz, 2H), 3.33-3.29 (m, 4H), 2.42 (t, J=6.9 Hz, 2H), 2.19 (s, 6H);ESI-MS (m/z, %): 331 (MH⁺, 100), 260 (25); ESI-HRMS calculated forC₁₇H₂₃N₄OS (MH⁺): 331.1587, Observed: 331.1594; HPLC purity: 99.0% byarea.

Example 7 Synthesis ofN-(4-(2-(ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamidedihydrochloride (7)

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine

For complete experimental details and spectral data, see Example 4.

2-Chloro-1-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanone

A suspension of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine (8 g, 44.4mmol) in anhydrous toluene (100 mL) under positive argon pressure wastreated dropwise with 2-chloroacetyl chloride (3.91 mL, 48.8 mmol). Themixture was heated at reflux for 25 minutes and became a bright yellowsolution. The solution was allowed to cool to room temperature, and asaturated NaHCO₃ solution (150 mL) was added. The mixture wastransferred to a separatory funnel, and the product was extracted withCH₂Cl₂ (2×200 mL). The combined organic layers were filtered through apad of silica gel, washed with CH₂Cl₂ (50 mL), and concentrated to givea yellow solid (10 g, 88%). ¹H-NMR (DMSO-d₆) δ 8.22 (d, J=9.0 Hz, 1H),7.80 (dd, J=2.7, 9.3 Hz, 1H), 7.72 (d, J=2.7 Hz, 1H), 4.74 (s, 2H), 4.40(t, J=4.2 Hz, 2H), 3.94 (t, J=4.8 Hz, 2H); ESI-MS (m/z, %): 279 (MNa⁺,30), 257 (MH⁺, 100).

2-(Ethylamino)-1-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanone

A solution of2-chloro-1-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanone (9.9 g,38.6 mmol) in anhydrous acetonitrile (250 mL) under positive argonpressure was treated with K₂CO₃ (42.7 g, 309 mmol) to give a yellowsuspension. The mixture was cooled to 0° C. and treated with ethanaminehydrochloride (15.7 g, 193 mmol). The reaction was brought to roomtemperature and stirred for 2.5 hours. The mixture was then filteredthrough a pad of Celite, washed with CH₂Cl₂ (500 mL), and concentrated.The residue was subjected to flash chromatography on silica gel usingEtOAc, followed by 2.5:97.5 (2M NH₃ in MeOH):CH₂Cl₂ to give a yellowsolid (7.73 g, 76%). ¹H-NMR (DMSO-d₆) δ 8.29 (d, J=9.0 Hz, 1H), 7.80(dd, J=2.7, 9.3 Hz, 1H), 7.71 (d, J=2.7 Hz, 1H), 4.36 (t, J=4.2 Hz, 2H),3.93 (t, J=4.8 Hz, 2H), 3.71 (s, 2H), 2.62 (q, J=7.2 Hz, 2H), 1.05 (t,J=7.2 Hz, 3H). ESI-MS (m/z, %): 266.1 (MH⁺, 100).

tert-Butyl ethyl(2-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)-2-oxoethyl)carbamate

Under positive argon pressure, a solution of2-(ethylamino)-1-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanone (7.73g, 29.1 mmol), di-tert-butyl dicarbonate (6.68 g, 30.6 mmol), andtriethylamine (8.19 mL, 58.3 mmol) in anhydrous 1,4 dioxane (100 mL) wasstirred for half an hour at room temperature. The mixture was dilutedwith water (200 mL), and the product was extracted into CH₂Cl₂ (2×200mL). The combined organic layers were washed with brine (100 mL), dried(Na₂SO₄), and concentrated. The residue was subjected to a short columnof silica gel using CH₂Cl₂ followed by 2.5:97.5 (2M NH₃ in MeOH):CH₂Cl₂to give a solid (8.83 g, 83%). ¹H-NMR (DMSO-d₆) δ 8.30-8.22 (m, 1H),7.82-7.78 (m, 1H), 7.71-7.70 (m, 1H), 4.36-4.28 (m, 4H), 3.94-3.92 (m,2H), 3.24 (t, J=2.7 Hz, 2H), 1.40, 1.30 (2×s, 9H), 1.07-1.04 (m, 3H).ESI-MS (m/z, %): 388 (MNa⁺, 50), 366 (MH⁺, 35), 266 (100).

tert-Butylethyl(2-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate

A yellow solution of tert-butylethyl(2-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)-2-oxoethyl) carbamate(8.7 g, 23.8 mmol) in anhydrous THF (50 mL) under positive argonpressure was treated with borane-THF complex (1M in THF, 47.6 mL, 47.6mmol) to give an orange solution. The mixture was refluxed for 20minutes. It was then cooled in an ice bath and treated dropwise withMeOH (100 mL). The solution stirred for half an hour at roomtemperature, and the reaction was then concentrated. The residue wassubjected to flash chromatography on silica gel using 1:9 EtOAc:hexanesto give an orange solid (7.5 g, 91%). ¹H-NMR (DMSO-d₆) δ 7.72 (dd,J=2.7, 9.0 Hz, 1H), 7.49-7.47 (m, 1H), 6.85-6.83 (m, 1H), 4.18 (t, J=4.2Hz, 2H), 3.59-3.53 (m, 2H), 3.37-3.36 (m, 2H), 3.20-3.18 (m, 2H), 1.47(s, 2H), 1.30 (br s, 9H), 1.02 (t, J=6.9 Hz, 3H); ESI-MS (m/z, %): 374(MNa⁺, 86), 352 (MH⁺, 7.8), 296 (100), 252 (87).

tert-Butylethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate

A suspension of tert-butylethyl(2-(7-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate (7 g,19.92 mmol) and Pd—C (2.11 g, 1.992 mmol, 10% wt) in anhydrous EtOH (100mL) was purged with hydrogen gas. The reaction was stirred at roomtemperature for 16 hours under a hydrogen atmosphere (balloon pressure).The reaction mixture was filtered through a Celite pad and washed withEtOH (50 mL). The filtrate containing tert-butyl2-(7-amino-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl(ethyl)carbamate wastreated with methyl thiophene-2-carbimidothioate hydroiodide (11.36 g,39.8 mmol) and stirred overnight at room temperature. The reaction wasconcentrated and then partitioned between saturated NaHCO₃ solution (150mL) and CH₂Cl₂ (75 mL). The mixture was transferred to a separatoryfunnel, and the product was extracted into CH₂Cl₂ (2×100 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated. Theresidue was subjected to flash chromatography on silica gel using asequence of eluents (CH₂Cl₂; 1:99 (2M NH₃ in MeOH):CH₂Cl₂; 1.75:98.25(2M NH₃ in MeOH):CH₂Cl₂; and 2.5:97.5 (2M NH₃ in MeOH):CH₂Cl₂) to give abrown solid (4 g, 46.7%). ¹H-NMR (CDCl₃) δ 7.43-7.41 (m, 2H), 7.11-7.05(m, 1H), 6.72-6.69 (m, 1H), 6.54-6.51 (m, 2H), 4.23-4.20 (m, 2H),3.39-3.25 (m, 8H), 1.47 (s, 9H), 1.11-1.10 (m, 3H); ESI-MS (m/z, %): 431(MH⁺, 100).

N-(4-(2-(Ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide

Under positive argon pressure, an orange suspension of tert-butylethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate(4 g, 9.29 mmol) in MeOH (30 mL) and 1N HCl (30 mL) was refluxed for 1hour. The mixture was then cooled to room temperature, transferred to anice bath, and 1N NaOH (75 mL) was then added. The solution turned amilky green colour with a black suspension. The mixture was then treatedwith CH₂Cl₂ (50 mL) and stirred for half an hour. After this time, themixture was transferred to a separatory funnel and the product wasextracted into CH₂Cl₂ (2×50 mL). The combined organic layers were dried(Na₂SO₄) and concentrated. The residue was subjected to flashchromatography on silica gel using the following sequence of eluents:EtOAc; 1:99 (2M NH₃ in MeOH):CH₂Cl₂; 2.5:97.5 (2M NH₃ in MeOH):CH₂Cl₂;and 5:95 (2M NH₃ in MeOH):CH₂Cl₂. A brown solid was obtained (2 g,65.1%). ¹H-NMR (CDCl₃) δ 7.40-7.37 (m, 2H), 7.07-7.04 (m, 1H), 6.75-6.72(m, 1H), 6.52-6.50 (m, 2H), 4.82 (brs, 2H), 4.24 (t, J=4.5 Hz, 2H),3.39-3.31 (m, 4H), 2.86 (t, J=6.3 Hz, 2H), 2.71 (q, J=6.9 Hz, 2H), 1.13(t, J=7.2 Hz, 3H); ESI-MS (m/z, %): 331 (MH⁺, 100), 260 (95); HPLCpurity: 94.1% by area.

N-(4-(2-(Ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamidedihydrochloride (7)

A suspension ofN-(4-(2-(ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)thiophene-2-carboximidamide(0.1 g, 0.303 mmol) in MeOH (3 mL), under positive argon pressure, wastreated with a 1M HCl ethereal solution (1.51 mL, 1.51 mmol). Thereaction stirred at room temperature for 1 hour, and the mixture wasthen concentrated to give a yellow-brown solid (93 mg, 76%). ¹H-NMR(DMSO-d₆) δ 11.20 (brs, 1H), 9.67 (brs, 1H), 9.23 (brs, 2H), 8.67 (brs,1H), 8.16-8.08 (m, 2H), 7.37-7.36 (m, 1H), 7.04 (d, J=8.4, 1H),6.87-6.81 (m, 2H), 4.30-425 (m, 2H), 3.66 (t, J=6.3 Hz, 2H), 3.12-2.95(m, 4H), 2.58-2.45 (m, 2H), 1.24 (t, J=7.2 Hz, 3H); ESI-MS (m/z, %): 331(MH⁺, free base, 100), 260 (95); ESI-HRMS calculated for C₁₉H₂₅N₄OS(MH⁺, free base): 331.1587, observed: 331.1597; HPLC purity: 95.4% byarea.

Example 8 Synthesis ofN-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(8)

2-Amino-5-nitrobenzenethiol

To a stirred suspension of 6-nitrobenzo[d]thiazole (1.64 g, 9.10 mmol)in ethanol (16 mL) was added hydrazine hydrate (2.66 mL, 54.6 mmol). Theresulting dark solution was stirred overnight at room temperature. Themixture was then diluted with water and slowly acidified with 3 M HCl,giving a yellow suspension. The mixture was then extracted withdichloromethane. The combined organics were dried, filtered, andconcentrated, giving a yellow solid, used directly in the subsequentreaction. ¹H NMR (DMSO-d₆) δ 8.09 (brs, 1H), 7.87 (d, J=8.7 Hz, 1H),6.74 (d, J=9.0 Hz, 1H), 6.27 (brs, 2H); MS-EI: (m/z, %) 170 (M+, 100),140 (28), 124 (35).

7-Nitro-2H-benzo[b][1,4]thiazin-3(4H)-one

To a stirred solution of 2-amino-5-nitrobenzenethiol (1.72 g, 10.11mmol) in tetrahydrofuran (10 mL) was added sodium bicarbonate (2.80 g,33.4 mmol) as a solution in water (40.0 mL). To this dark red solutionwas added 2-chloroacetyl chloride (0.885 mL, 11.12 mmol). The resultingmixture, which turned faint red, was then stirred at room temperatureovernight. The mixture was then diluted with water and extracted threetime with dichloromethane. The combined organics were dried, filtered,concentrated, and then chromatographed in 10% ethyl acetate indichloromethane, giving the desired product (1.02 g, 48.0%). ¹H NMR(DMSO-d₆) δ 11.17 (s, 1H), 8.24 (d, J=2.7 Hz, 1H), 8.07 (dd, J=9.0, 2.7Hz, 1H), 7.13 (d, J=9.0 Hz, 1H), 3.60 (s, 2H); MS-EI: (m/z, %) 210 (M+,100), 181 (40), 131 (46).

4-(2-(Dimethylamino)ethyl)-7-nitro-2H-benzo[b][1,4]thiazin-3(4H)-one

To a stirred suspension of 7-nitro-2H-benzo[b][1,4]thiazin-3(4H)-one(1.02 g, 4.85 mmol) and potassium carbonate (4.69 g, 34.0 mmol) in apressure flask in DMF (30 mL) under argon was added2-chloro-N,N-dimethylethanamine hydrochloride (3.49 g, 24.26 mmol). Theflask was immediately sealed and heated with stirring to 90° C.overnight. The mixture was then cooled to room temperature, diluted withwater and extracted (2× ethyl acetate). The combined organics were thenwashed with a 1:1 mixture of brine and water (3×) then brine (1×). Theorganic phase was dried, filtered, concentrated, and thenchromatographed in 5% (2M NH₃ in MeOH) in dichloromethane, giving thedesired product (755 mg, 55.3%). ¹H NMR (DMSO-d₆) δ 8.30 (d, J=2.7 Hz,1H), 8.13 (dd, J=9.3, 2.7 Hz, 1H), 7.59 (d, J=9.3 Hz, 1H), 4.11 (t,J=6.9 Hz, 2H), 3.62 (s, 2H), 2.41 (t, J=6.9 Hz, 2H), 2.17 (s, 6H);MS-EI: (m/z, %) 281 (M+, 55), 251 (65), 58 (100).

N,N-Dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

To a stirred solution of4-(2-(dimethylamino)ethyl)-7-nitro-2H-benzo[b][1,4]thiazin-3(4H)-one(730 mg, 2.59 mmol) in tetrahydrofuran (3 mL) under argon was addedborane (1M in THF; 7.784 mL, 7.78 mmol). The resulting mixture wasstirred overnight at room temperature, producing a yellow precipitate.The mixture was heated briefly to 60° C. to break up the solid. Thereaction was then cooled to 0° C. and quenched with methanol (slowly,until bubbling ceased). The mixture was then concentrated, redissolvedin MeOH (15 mL) and 1 M HCl (5 mL), and heated at 60° C. for 30 minutes.The quenched mixture was then diluted with water and sodium carbonate,and then extracted with dichloromethane (3×). The combined organics weredried, filtered, concentrated, and then chromatographed in ethylacetate, giving a yellow solid, and the desired complex as a boranecomplex, which was used directly in the subsequent reaction.

To a stirred solution ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine-boranecomplex (350 mg, 1.245 mmol) in methanol (5 mL) and THF (10 mL) underargon was added 10% Pd—C (132 mg, 0.124 mmol). The mixture was thenstirred overnight at room temperature. The suspension was then filteredthrough a pad of Celite, concentrated, and chromatographed in 5% (2M NH₃in MeOH) in dichloromethane, giving the desired product (106.6 mg,32.0%). ¹H NMR (DMSO-d₆) δ 7.85-7.81 (m, 2H), 6.81 (d, J=9.0 Hz, 1H),3.81 (t, J=5.1 Hz, 2H), 3.56 (t, J=6.9 Hz, 2H), 3.06 (t, J=5.1 Hz, 1H),2.47 (t, J=6.9 Hz, 2H), 2.21 (s, 6H); MS-EI: (m/z, %) 267 (M+, 13), 179(29), 58 (100).

Phenylmethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine (100mg, 0.374 mmol) in dichloromethane (5 mL) under argon was added phenylcarbonochloridate (0.094 mL, 0.748 mmol). The resulting mixture wasstirred at room temperature. A precipitate was observed, andtriethylamine (3 drops) was added to aid dissolution (reaction turnedclear). The mixture was then stirred over the weekend. The clear yellowmixture was then diluted with water and sodium carbonate and thenextracted with dichloromethane (3×). The combined organics were dried,filtered, concentrated, and then chromatographed in ethyl acetate,giving the desired product (139 mg, 100%). ¹H NMR (DMSO-d₆) δ 7.84-7.74(m, 2H), 7.39-7.31 (m, 2H), 7.23-7.16 (m, 1H), 7.03-6.93 (m, 3H),3.85-3.78 (m, 3H), 3.73-3.65 (m, 2H), 3.55-3.50 (m, 1H), 3.09, 2.97 (2s,3H), 3.09-3.05 (m, 2H); MS-EI: (m/z, %) 373 (M+, 4), 209 (31), 179(100), 151 (28).

Phenyl2-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(methyl)carbamate

To a stirred solution of phenyl methyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate (100 mg,0.268 mmol) in tetrahydrofuran (4 mL) and ethanol (4.00 mL) under argonwas added 10% Pd—C (28.5 mg, 0.027 mmol). The resulting mixture wasstirred at room temperature overnight under an atmosphere of hydrogen(balloon pressure). The mixture was then filtered through a pad ofCelite and concentrated, giving the desired product (81 mg, 88%) as adark oil. MS-EI: (m/z, %) 343 (M+, 64), 179 (100), 151 (45).

Phenylmethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution of phenyl2-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(methyl)carbamate (80mg, 0.233 mmol) in ethanol (5 mL) under argon was added methylthiophene-2-carbimidothioate hydroiodide (133 mg, 0.466 mmol). Themixture was then stirred overnight at room temperature. The mixture wasthen diluted with water and sodium carbonate and then extracted withdichloromethane (2×). The combined organics were dried, filtered,concentrated, and then chromatographed in ethyl acetate, yielding thedesired product (72 mg, 68%). ¹H NMR (DMSO-d₆) δ 7.74-7.69 (m, 1H),7.58-7.56 (m, 1H), 7.42-7.34 (m, 2H), 7.25-7.17 (m, 1H), 7.14-7.05 (m,3H), 6.83-6.80 (m, 1H), 6.53-6.44 (m, 2H), 6.36 (brs, 2H), 3.63-3.55 (m,4H), 3.49-3.45 (m, 2H), 3.11, 2.98 (2s, 3H), 3.06-3.01 (m, 2H); MS-EI:(m/z, %) 452 (M+, 97), 288 (100).

N-(4-(2-(Methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(8)

To a stirred solution of phenylmethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(65 mg, 0.144 mmol) in ethanol (4 mL) was added sodium hydroxide (57.4mg, 1.436 mmol) as a solution in water (2 mL). The resulting mixture washeated to 78° C. and stirred overnight. The mixture was then cooled toroom temperature, diluted with water and sodium hydroxide, and extractedwith dichloromethane (5×). The combined organics were dried, filtered,concentrated, and then chromatographed in 10% (2M NH₃ in MeOH) indichloromethane, giving the desired product (22 mg, 46%). ¹H NMR(DMSO-d₆) δ 7.69 (d, J=3.3 Hz, 1H), 7.56 (d, J=5.1 Hz, 1H), 7.07 (t,J=4.4 Hz, 1H), 6.72 (d, J=8.7 Hz, 1H), 6.53-6.44 (m, 2H), 6.29 (brs,2H), 3.54-3.51 (m, 2H), 3.3 (m, 2H), 3.04-3.00 (m, 2H), 2.66 (t, J=6.8Hz, 2H), 2.32 (s, 3H); MS-EI: (m/z, %) 332 (M+, 75), 288 (97), 260(100); EI-HRMS calculated for C₁₆H₂₀N₄S₂ (M+), calculated: 332.1129,observed: 332.1145. HPLC: 95% by area.

Example 9 Synthesis ofN-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-thiophene-2-carboximidamide(9)

N,N-Dimethyl-2-(6-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanamine

A solution of 6-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine (1.0 g, 5.55mmol) in anhydrous DMF (25 mL) under argon was cooled and treatedportionwise with sodium hydride (0.677 g, 16.93 mmol) at 5-10° C.2-chloro-N,N-dimethylethanamine hydrochloride (1.599 g, 11.10 mmol) wasadded portionwise at 5-10° C., and the resulting heterogeneous red-brownmixture was allowed to warm to room temperature. After 3 hours at roomtemperature, the mixture was heated to 90° C. and stirred for 30minutes. The mixture was cooled to room temperature, quenched by theaddition of water (25 mL), and diluted with EtOAc. A small amount of 1 NNaOH was added to basify the mixture to pH ˜9-10. The organic layer wasseparated, and the aqueous layer was further extracted with EtOAc. Thecombined organic layers were washed with brine (×2), dried (Na₂SO₄),filtered, and concentrated to afford a dark red oil. The material waspurified on silica gel, eluting with 5% MeOH/CH₂Cl₂, to yield the titlecompound as a dark orange-red oil (458 mg, 32.8%). ¹H NMR (DMSO-d₆) δ7.49-7.39 (m, 2H), 6.87-6.84 (m, 1H), 4.27-4.25 (m, 2H), 3.50-3.37 (m,4H), 2.43 (t, 2H, J=6.7 Hz), 2.19 (s, 6H); ESI-MS (m/z, %): 252 (MH⁺,100).

4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-amine

To an oven dried, argon purged 100 mL round bottom flask fitted with amagnetic stirbar was addedN,N-dimethyl-2-(6-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethanamine (0.10g, 0.398 mmol) and EtOH (10 mL). At this time, stirring of the reactionbegan. 10% Pd—C (0.042 g, 0.040 mmol) was added, the flask wasevacuated, and the mixture was subjected to hydrogenation under standardconditions (atmospheric H₂ pressure using a balloon). After 3 hours, themixture was filtered through a pad of Celite and washed with ethanol.The pale peach colored solution, which darkened upon exposure to air,was concentrated to approximately 10 mL to yield the title compound.This compound was utilized immediately in the next step. ESI-MS (m/z,%): 222 (MH⁺, 100).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)thiophene-2-carboximidamide

To a stirred ethanolic solution of4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-amine(0.088 g, 0.398 mmol) under an argon atmosphere was added methylthiophene-2-carbimidothioate hydroiodide (0.227 g, 0.795 mmol). Theresulting faintly cloudy yellow mixture stirred overnight at roomtemperature. After 20 hours, argon was bubbled through the mixture,which was then concentrated to afford a residue. The residue was thenpartitioned between EtOAc (100 mL) and saturated NaHCO₃ (˜10 mL)solution. The aqueous layer (pH˜9) was further extracted with EtOAc, andthe combined organic layers were washed with brine, dried (Na₂SO₄),filtered, and concentrated to yield a yellow residue. Purification onsilica gel (dry chromatography, eluting with 5-7.5% (2M NH₃MeOH)/CH₂Cl₂)yielded the title compound 9 as a yellow residue (70 mg, 53.3%). ¹H NMR(DMSO-d₆) δ 7.70 (d, 1H, J=2.7 Hz), 7.57 (d, 111, J=5.0 Hz), 7.07 (dd,1H, J=5.0, 3.7 Hz), 6.59 (d, 1H, J=8.2 Hz), 6.30 (brs, 2H), 6.14 (s,1H), 6.00 (d, 1H, J=8.1 Hz), 4.18-4.02 (m, 2H), 3.41-3.25 (m, 4H+H₂O),2.41 (t, 2H, J=6.7 Hz), 2.17 (s, 6H); ESI-MS (m/z, %): 331 (MH⁺, 100).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)thiophene-2-carboximidamidedihydrochloride

N-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)thiophene-2-carboximidamide(0.065 g, 0.197 mmol) was dissolved in anhydrous MeOH (5 mL) and treatedwith hydrogen chloride (1M in diethyl ether; 0.433 mL, 0.433 mmol) for 5minutes at room temperature. The reaction was then concentrated to yieldthe title compound as a yellow solid (58.5 mg, 73.7%). ¹H NMR (DMSO-d₆)δ 11.36 (brs, 1H), 11.02 (brs, 1H), 9.70 (brs, 1H), 8.69 (brs, 1H), 8.15(app d, 2H, J=4.5 Hz), 7.37 (app t, 1H, J=4.3 Hz), 7.07 (s, 1H), 6.84(d, 1H, J=8.3 Hz), 6.62 (d, 1H, J=8.4 Hz), 4.31-4.18 (m, 2H), 3.76-3.65(m, 2H), 3.44-3.34 (m, 2H), 3.35-3.22 (m, 2H), 2.78, 2.77 (2×s, 6H);ESI-MS (m/z, %): 331 (MH⁺, free base, 100); ESI-HRMS calculated forC₁₇H₂₃N₄OS (MH⁺, free base): 331.1587; observed: 331.1579.

Example 10 Synthesis ofN-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-thiophene-2-carboximidamidedihydrochloride (10)

tert-Butylmethyl(2-(6-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate

To an oven dried, argon purged flask fitted with a magnetic stirbar wasadded tert-butyl methyl(2-oxoethyl)carbamate (0.135 g, 0.777 mmol), andDCE (10 mL). At this time, stirring began, and6-nitro-3,4-dihydro-2H-benzo[b][1,4]oxazine (0.10 g, 0.555 mmol) wasadded, followed by acetic acid (0.064 mL, 1.110 mmol) and sodiumtriacetoxyborohydride (0.294 g, 1.388 mmol). The resulting mixturestirred overnight at room temperature. After 18 hours, the reaction wasquenched by the addition of saturated NaHCO₃ (10 mL) and extracted withCH₂Cl₂. The aqueous layer was extracted further, and the combinedorganics were washed with brine, dried (Na₂SO₄), and concentrated. Thecrude product was combined with the product of a second reaction(prepared in accordance with the above procedure, with the omission ofacetic acid). The combined products were purified on silica gel elutingwith 50-70% EtOAc/hexanes to yield the title compound as a yellowresidue (130 mg, 34.7%). ¹H NMR (DMSO-d₆) δ 7.58-7.37 (m, 2H), 6.92-6.80(m, 1H), 4.25 (t, 2H, J=4.2 Hz), 3.58-3.36 (m, 6H), 2.87-2.74 (m, 3H),1.27, 1.18 (2×s, 9H); ESI-MS (m/z, %): 360 (MNa⁺, 100).

tert-Butyl2-(6-amino-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl(methyl)carbamate

To an oven dried, argon purged round bottom flask fitted with a magneticstirbar was added tert-butyl methyl(2-(6-nitro-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate (0.13 g,0.385 mmol), EtOH (10 mL). At this time, stirring began. 10% Pd—C (0.041g, 0.039 mmol) was added, the flask evacuated, and the material wassubjected to hydrogenation under standard conditions as describedherein. After 2.5 hours, the mixture was filtered through a pad ofCelite and washed with ethanol. The pale peach colored solution, whichdarkened slowly upon exposure to air, was concentrated to a volume of˜10 mL to yield the title compound which was utilized immediately in thenext step. A small portion was concentrated to dryness for analyticalpurposes. ¹H NMR (DMSO-d₆) δ 6.34 (d, 1H, J=8.3 Hz), 6.03-5.95 (m, 1H),5.75 (dd, 1H, J=8.3, 2.3 Hz), 4.35 (m, 2H), 3.98 (t, 2H, J=4.1 Hz),3.30-3.15 (m, 6H), 2.81 (s, 3H), 1.38, 1.30 (2×s, 9H); ESI-MS (m/z, %):308 (MH⁺, 90), 252 (100).

tert-Butylmethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]-oxazin-4(3H)-yl)ethyl)carbamate

To a stirred ethanolic solution of tert-butyl2-(6-amino-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl(methyl)carbamate (0.118g, 0.384 mmol) under an argon atmosphere was charged methylthiophene-2-carbimidothioate hydroiodide (0.219 g, 0.768 mmol), and theresulting yellow solution stirred at room temperature. After 44 hours,argon was bubbled through the mixture, which was then concentrated toresidue and partitioned between EtOAc (100 mL) and saturated NaHCO₃solution (10 mL). The aqueous layer (pH˜9) was further extracted withEtOAc, and the combined organic layers were washed with brine, dried(Na₂SO₄), filtered, and concentrated to yield an orange residue. Theresidue was purified on silica gel twice, with the first purificationusing 100% EtOAc as the eluent and the second purification using 5% (2MNH₃ in MeOH)/CH₂Cl₂ to yield the title compound as a yellow solid (80mg, 50.0%). ¹H NMR (DMSO-d₆) δ 7.68 (d, 1H, J=2.9 Hz), 7.57 (d, 1H,J=5.0 Hz), 7.10-7.03 (m, 1H), 6.60 (d, 1H, J=8.1 Hz), 6.30 (m, 3H), 6.00(dd, 1H, J=8.1, 1.6 Hz), 4.18-4.02 (m, 2H), 3.40-3.21 (m, 6H, mergedwith H₂O peak), 2.81 (s, 3H), 1.34, 1.29 (2×s, 9H); ESI-MS (m/z, %): 417(MH⁺, 100).

N-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-thiophene-2-carboximidamidedihydrochloride

To a solution of tert-butylmethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]oxazin-4(3H)-yl)ethyl)carbamate(0.07 g, 0.168 mmol) in HPLC grade MeOH (10 mL) was added 3Mhydrochloric acid (0.56 mL, 1.681 mmol). The resulting yellow solutionwas heated to reflux under an atmosphere of argon. After 60 minutes, thereaction was cooled to room temperature and concentrated. The residuewas diluted with H₂O (20 mL) and rinsed sequentially with CH₂Cl₂ (20 mL)and EtOAc (20 mL). The aqueous layer was concentrated and dried to yieldthe title compound 10 as a yellow solid (37 mg, 56.5%). ¹H NMR (DMSO-d₆)δ 11.37 (brs, 1H), 9.72 (brs, 1H), 9.23 (brs, 2H), 8.66 (brs, 1H),8.21-8.08 (m, 2H), 7.37 (app t, 1H, J=4.4 Hz), 7.03 (s, 1H), 6.84 (d,1H, J=8.3 Hz), 6.60 (d, 1H, J=7.3 Hz), 4.31-4.20 (m, 2H), 3.70-3.45 (m,2H, merged with H₂O peak), 3.40-3.34 (m, 2H), 3.18-3.02 (m, 2H),2.60-2.50 (m, 3H); ESI-MS (m/z, %): 317 (MH⁺, free base, 100); ESI-HRMScalculated for C₁₆H₂₁N₄OS (MH⁺, free base): 317.1430; observed:317.1422.

Example 11 Synthesis ofN-(4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(11)

2-Amino-5-nitrobenzenethiol

To a suspension of 6-nitrobenzo[d]thiazole (5 g, 27.7 mmol) in ethanol(45 mL) was added hydrazine hydrate (8.10 mL, 166 mmol). The resultingmixture (dark red) was stirred overnight at room temperature. Themixture was then acidified by the slow, careful addition of 3M HCl inwater (50 mL). This mixture was further diluted with water (50 mL), andthe resulting yellow/orange precipitate was extracted withdichloromethane (3×). The combined organics were dried, filtered, andconcentrated, giving an orange solid (4.62 g, 98%). ¹H NMR (DMSO-d₆) δ8.12-8.09 (m, 1H), 7.88-7.85 (m, 1H), 6.75-6.71 (m, 1H), 6.28 (brs, 2H);ESI-MS (m/z, %): 170 (MH⁺, 100).

2-(2-Chloroethylthio)-4-nitroaniline

To a stirred suspension of 2-amino-5-nitrobenzenethiol (4.11 g, 24.15mmol) in ethanol (50 mL) and water (10 mL) was added sodium hydroxide(0.966 g, 24.15 mmol). To the resulting solution was added1-bromo-2-chloroethane (8.04 mL, 97 mmol), and the resulting mixture wasstirred at room temperature for 3 hours. The mixture was then dilutedwith ethyl acetate and washed with saturated sodium carbonate (3×). Theorganic phase was dried, filtered, and concentrated, giving a darkyellow solid (5.13 g, 91%). ¹H NMR (DMSO-d₆) δ 8.16 (d, J=2.7 Hz, 1H),7.97 (dd, J=9.0, 2.7 Hz, 1H), 6.94 (brs, 2H), 6.79 (d, J=9.0 Hz, 1H),3.68 (t, J=7.2 Hz, 2H), 3.11 (t, J=7.2 Hz, 2H); ESI-MS (m/z, %): 233(MH⁺, 100), 216 (79).

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

To a stirred solution of 2-(2-chloroethylthio)-4-nitroaniline (5.13 g,22.05 mmol) in DMF (70 mL) was added potassium carbonate (9.14 g, 66.1mmol), followed by sodium iodide (0.330 g, 2.205 mmol). The resultingsuspension was stirred at room temperature overnight. The mixture wasthen diluted with water, and the resulting crystals were collected byvacuum filtration, giving the desired product (4.06 g, 94%). ¹H NMR(DMSO-d₆) δ 7.81-7.73 (m, 3H), 6.58 (t, J=9.0 Hz, 1H), 3.65-3.60 (m,2H), 3.01-2.97 (m, 2H); ESI-MS (m/z, %): 197 (MH⁺, 100), 180 (99).

7-Nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine

A mixture of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (1 g, 5.10mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (1.734 g, 10.19 mmol),and nBu₄NBr (0.082 g, 0.255 mmol) in CH₂Cl₂ (10 mL) and 50% NaOHsolution (10 mL) was stirred at room temperature for 16 hours. Themixture was diluted with CH₂Cl₂ (25 mL) and water (50 mL). The mixturewas transferred to a separatory funnel, and the layers were separated.The aqueous layer was extracted in CH₂Cl₂ (2×25 mL). The combinedorganic layers were dried (Na₂SO₄), filtered, and concentrated. The redcrude material was subjected to flash chromatography on silica gel(CH₂Cl₂, then 1.75-5% (2M NH₃ in MeOH)/CH₂Cl₂) to give7-nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazineas a brown viscous oil (0.63 g, 42%). ¹H NMR (DMSO-d₆) δ 7.86-7.82 (m,2H), 6.82 (d, J=9.0 Hz, 1H), 3.81 (t, J=4.8 Hz, 2H), 3.58 (t, J=6.9 Hz,2H), 3.33 (s, 4H), 3.05 (t, J=5.1 Hz, 2H), 2.63 (t, J=6.9 Hz, 2H), 2.50(s, 4H), 1.67 (s, 4H); ESI-MS (m/z, %): 296, 294 (MH⁺, 100), 98 (13).

4-(2-(Pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

To a solution of7-nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine(0.637 g, 2.172 mmol) in MeOH (30 mL) was added Raney-Nickel (slurry inwater) (˜30 mg, 2.172 mmol) followed by hydrazine hydrate (1.056 mL,21.72 mmol), and the mixture was immersed in a preheated oil bath andrefluxed for 1 hour. The solution was cooled to room temperature,filtered through a pad of Celite, and washed with 100 mL of MeOH. Thecrude material was filtered through a silica plug (3.5-10% 2M NH₃ inMeOH/CH₂Cl₂). The solvent was evaporated to give the title product as adark brown oil (0.61 g, quantitative). ¹H NMR (DMSO-d₆) δ 6.49 (d, J=8.7Hz, 1H), 6.26-6.23 (m, 2H), 4.42 (brs, 2H), 3.38-3.35 (m, 2H), 3.21 (t,J=7.2 Hz, 2H), 2.97-2.94 (m, 2H), 2.55 (t, J=7.2 Hz, 2H), 2.45 (brs,4H), 1.66 (brs, 4H); ESI-MS (m/z, %): 266, 264 (MH⁺, 100), 98 (34).

N-(4-(2-(Pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

Methyl thiophene-2-carbimidothioate hydroiodide (1.239 g, 4.34 mmol) wasadded to a mixture of4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.572 g, 2.172 mmol) in EtOH (20 mL). The reaction was stirredovernight at room temperature. The reaction was quenched with saturatedsodium bicarbonate solution (30 mL). The mixture was then extracted withCH₂Cl₂ (50 mL), and the aqueous phase was washed with CH₂Cl₂ (50 mL).The combined organic fractions were washed with brine (50 mL) and dried(Na₂SO₄). The crude material was subject to flash chromatography onsilica gel (CH₂Cl₂ then 5% (2M NH₃MeOH)/CH₂Cl₂). The collected fractionsgave compound 11 as light brown viscous liquid (0.71 g, 88%). ¹H NMR(DMSO-d₆) δ 7.69 (d, J=3.6 Hz, 1H), 7.56 (d, J=5.1 Hz, 1H), 7.08-7.06(m, 1H), 6.67 (t, J=8.7 Hz, 1H), 6.50 (d, J=8.4 Hz, 1H), 6.46 (s, 1H),6.32 (brs, 2H), 3.57-3.53 (m, 2H), 3.39-3.34 (m, 2H), 3.03-3.00 (m, 2H),2.60 (t, J=7.2 Hz, 2H), 2.50 (brs, 4H), 1.68 (brs, 4H); ESI-MS (m/z, %):375, 373 (MH⁺, 47), 278, 276 (100), 98 (21).

N-(4-(2-(Pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

To a stirred solution ofN-(4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.3524 g, 0.946 mmol) in MeOH (2 mL) was added 1M HCl in ether (4.73mL, 4.73 mmol) at room temperature. The mixture was stirred for 5minutes under argon atmosphere and was then concentrated to give ayellow foam (0.646 g, quantitative). ¹H NMR (DMSO-d₆) δ 11.51 (s, 1H),11.23 (s, 1H), 9.68 (s, 1H), 8.69 (s, 1H), 8.15-8.11 (m, 2H), 7.37-7.34(m, 1H), 7.08 (d, J=9.3 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 3.81-3.76 (m,2H), 3.66-3.64 (m, 2H), 3.56-3.46 (m, 2H), 3.33-3.30 (m, 2H), 3.13-3.10(m, 2H), 3.06-3.00 (m, 2H), 2.00-1.86 (m, 4H); ESI-MS (m/z, %): 375, 373(MH⁺, free base, 66), 278, 276 (100), 98 (21); HRMS (C₁₉H₂₅N₄S₂, MH⁺,Free base): calculated: 373.1515, observed: 373.1518. HPLC: 98% by area.

Example 12 Synthesis ofN-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride (12)

N,N-Dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

Prepared according to the procedure reported in Example 8.

4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

A dark mixture ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine (1.0g, 3.74 mmol) in methanol (10 mL) under an argon atmosphere was treatedwith Raney Nickel (˜0.22 g, 3.74 mmol) and hydrazine hydrate (1.82 mL,37.4 mmol). The reaction was transferred to a pre-heated oil bath at 65°C. After 50 minutes, the mixture was allowed to cool to room temperatureand then poured over a pad of Celite. The Celite pad was rinsed withmethanol (20 mL). The filtrate was concentrated, and the crude materialwas subjected to a short column on silica gel (1:9 (2M NH₃ inMeOH):CH₂Cl₂). The resulting brown residue was carried on to the nextstep. ¹H-NMR (DMSO-d₆) δ 6.49 (d, J=9.0 Hz, 1H), 6.27-6.23 (m, 2H), 4.42(brs, 2H), 3.40-3.30 (m, 2H), 3.18 (t, J=7.2 Hz, 2H), 2.98-2.94 (m, 2H),2.36 (t, J=7.2 Hz, 2H), 2.16 (s, 6H); ESI-MS (m/z, %): 238 (MH⁺, 100),193 (56), 147 (58).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A suspension of4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.888 g, 3.74 mmol) and methyl thiophene-2-carbimidothioate hydroiodide(2.134 g, 7.48 mmol) in dry ethanol (15 mL) was stirred at roomtemperature overnight (16 hours). The solvent was evaporated, and theresidue was partitioned between saturated NaHCO₃ solution (50 mL) andCH₂Cl₂ (25 mL). After stirring for 20 minutes, the mixture wastransferred to a separatory funnel, and the organic layer was removed.The aqueous layer was extracted into CH₂Cl₂ (2×25 mL). The combinedorganic layer was dried (Na₂SO₄), filtered, and concentrated. Theresidue was subjected to column chromatography using silica gel (CH₂Cl₂,then 5:95 (2M NH₃ in MeOH):CH₂Cl₂) to give the title product 12 as asolid (0.44 g, 33.9%). ¹H-NMR (DMSO-d₆) δ 7.69 (d, J=3.3 Hz, 1H), 7.56(d, J=5.1 Hz, 1H), 7.07 (dd, J=4.8, 3.9 Hz, 1H), 6.66 (d, J=8.7 Hz, 1H),6.52-6.46 (m, 2H), 6.32 (brs, 2H), 3.56-3.52 (m, 2H), 3.36-3.32 (m, 2H),3.03-3.00 (m, 2H), 2.41 (t, J=7.2 Hz, 2H), 2.19 (s, 6H); ESI-MS (m/z,%): 347 (MH⁺, 100), 276 (84%).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.44 g, 1.270 mmol) in dry ethanol (10 mL) was treated withhydrochloric acid (1M in ether; 6.35 mL, 6.35 mmol) and stirred at roomtemperature for 1 hour. The precipitate was isolated and washed withether to give a yellow-green powder. The powder was dried under reducedpressure. (0.46 g, 86%). ¹H-NMR (DMSO-d₆) δ 11.25 (brs, 2H), 9.69 (brs,1H), 8.69 (brs, 1H), 8.15-8.13 (m, 2H), 7.37-7.37 (m, 1H), 7.07-7.01 (m,3H), 3.82-3.77 (m, 2H), 3.70-3.60 (m, 2H), 3.25-3.21 (m, 2H), 3.15-3.10(m, 2H), 2.80 (d, J=3.6 Hz, 6H); ESI-MS (m/z, %): 347 (MH⁺, free base,100); ESI-HRMS calculated for C₁₇H₂₃N₄S₂ (MH⁺, free base): 347.1358,observed: 347.1343; HPLC purity: 97% by area.

Example 13 Synthesis ofN-(4-(2-(1-methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(13)

7-Nitro-3,4-dihydro-21′-benzo[b][1,4]thiazine

Prepared according to the procedure reported in Example 11.

4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

A mixture of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (1 g, 5.10mmol), 2-(2-chloroethyl)-1-methylpyrrolidine hydrochloride (1.876 g,10.19 mmol), and tetrabutylammonium bromide (0.082 g, 0.255 mmol) indichloromethane (10 mL) and 50% NaOH solution (10 mL) was stirred atroom temperature for 16 hours (overnight). The reaction was then dilutedwith dichloromethane (5 mL) and water (20 mL), and the mixture wastransferred to a separatory funnel. The organic layer was removed, andthe aqueous layer was extracted into dichloromethane (2×10 mL). Thecombined organic layers were dried (Na₂SO₄), filtered, and concentrated.The red crude material was subjected to column chromatography on silicagel using the Biotage purification system (80 g silicycle column;gradient: dichloromethane 3CV then ramp to 6:94 (2M NH₃ inmethanol):dichloromethane over 15CV; 254 nm; flow: 45 mL/min) to givethe title compound as yellow-orange solid (0.827 g, 52.8%). ¹H-NMR(DMSO-d₆) δ 7.87 (dd, J=3.0, 9.0 Hz, 1H), 7.83 (d, J=3.0 Hz, 1H), 6.79(d, J=9.3 Hz, 1H), 3.79-3.76 (m, 2H), 3.46 (t, J=8.1 Hz, 2H), 3.08-3.05(m, 2H), 2.99-2.92 (m, 1H), 2.23 (s, 3H), 2.10-2.06 (m, 2H), 1.92-1.78(m, 2H), 1.66-1.61 (m, 2H), 1.54-1.47 (m, 2H); ESI-MS (m/z, %): 308(MH⁺, 100).

4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

A solution of4-(2-(1-methylpyrrolidin-2-yl)ethyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine(0.82 g, 2.67 mmol) in methanol (10 mL) under an argon atmosphere wastreated with hydrazine hydrate (1.29 mL, 26.7 mmol) and a small amountof Raney Nickel (˜0.15 g, 2.67 mmol). The reaction was transferred to apre-heated oil bath at 65° C. After 3 hours, the reaction was pouredover a pad of Celite, rinsing with methanol. The filtrate wasconcentrated, and the residue was poured over a pad of silica gel. Thesilica gel pad was rinsed with 1:9 (2M NH₃ in methanol):dichloromethane(200 mL). The filtrate was concentrated, dried under reduced pressure,and carried on to the next step. ¹H-NMR (DMSO-d₆) δ 6.48 (brd, J=9.3 Hz,1H), 6.27-6.24 (m, 2H), 4.42 (brs, 2H), 3.33-3.29 (m, 2H), 3.13-3.06 (m,2H), 2.99-2.96 (m, 2H), 2.96-2.90 (m, 1H), 2.19 (s, 3H), 2.06-1.97 (m,2H), 1.96-1.78 (m, 2H), 1.63-1.60 (m, 2H), 1.56-1.40 (m, 2H); ESI-MS(m/z, %): 278 (MH⁺, 100).

N-(4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A suspension of4-(2-(1-methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.7 g, 2.52 mmol) and methyl thiophene-2-carbimidothioate hydroiodide(1.439 g, 5.05 mmol) in dry ethanol (15 mL) was stirred at roomtemperature overnight (16 hours) under an argon atmosphere. At thistime, the solvent was evaporated, and the residue was partitionedbetween saturated NaHCO₃ solution (50 mL) and dichloromethane (25 mL).The mixture was transferred to a separatory funnel, and the organiclayer was removed. The aqueous layer was extracted into dichloromethane(2×25 mL). The combined organic layers were dried (Na₂SO₄), filtered,and concentrated. The residue was subjected to column chromatography onsilica gel (sequence of eluents: 2:98 methanol:dichloromethane, 5:95methanol:dichloromethane, and 5:95 (2M ammonia inmethanol):dichloromethane) to give compound 13 as yellow syrup (0.48 g,49%). ¹H-NMR (DMSO-d₆) δ 7.69 (brd, J=2.7 Hz, 1H), 7.57 (brd, J=4.5 Hz,1H), 7.07 (dd, J=3.9, 4.8 Hz, 1H), 6.52 (dd, J=1.8, 8.1 Hz, 2H), 6.47(brd, J=1.8 Hz, 1H), 6.38 (brs, 2H), 3.51-3.47 (m, 2H), 3.28-3.22 (m,2H), 3.05-3.02 (m, 2H), 2.99-2.93 (m, 1H), 2.24 (s, 3H), 2.12-2.06 (m,2H), 1.94-1.87 (m, 2H), 1.68-1.63 (m, 2H), 1.51-1.42 (m, 2H); ESI-MS(m/z, %): 387 (MH⁺, 90), 276 (100), 194 (50).

N-(4-(2-(1-Methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(2-(1-methylpyrrolidin-2-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.48 g, 1.242 mmol) in dry ethanol (10 mL) was treated withhydrochloric acid (1M in ether; 6.21 mL, 6.21 mmol) and stirred for halfan hour. The precipitate was collected and rinsed with ether. Theprecipitate was dissolved in methanol and concentrated to give ayellow-orange crystalline solid (0.57 g, 100%). ¹H-NMR (DMSO-d₆) δ 11.22(brs, 1H), 11.17 (brs, 1H), 9.68 (brs, 1H), 8.72 (brs, 1H), 8.15-8.13(m, 2H), 7.37-7.34 (m, 1H), 7.05-6.99 (m, 2H), 6.91 (brd, J=8.7 Hz, 1H),3.66-3.64 (m, 2H), 3.55-3.41 (m, 3H), 3.38-3.22 (m, 1H), 3.12-3.09 (m,2H), 3.00-2.97 (m, 1H), 2.76 (d, J=4.8 Hz, 3H), 2.32-2.18 (m, 2H),1.99-1.86 (m, 3H), 1.77-1.71 (m, 1H); ESI-MS (m/z, %): 387 (MH⁺, freebase, 100), 194 (19); ESI-HRMS calculated for C₂₀H₂₇N₄S₂ (MH⁺, freebase): 387.1671, observed: 387.1654; HPLC purity: 97% by area.

Example 14 Synthesis ofN-(4-(2-(Piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(14)

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the procedure reported in Example 11.

7-Nitro-4-(2-(piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine

A mixture of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (1 g, 5.10mmol), 1-(2-chloroethyl)piperidine hydrochloride (1.876 g, 10.19 mmol),and tetrabutylammonium bromide (0.082 g, 0.255 mmol) in dichloromethane(10 mL) and 50% NaOH solution (10 mL) was stirred at room temperatureovernight (16 hours). The mixture was diluted with dichloromethane (5mL) and water (20 mL), and the mixture was then transferred to aseparatory funnel. The organic layer was removed, and the aqueous layerwas extracted in dichloromethane (2×10 mL). The combined organic layerswere dried (Na₂SO₄), filtered, and concentrated. The red crude materialwas subjected to column chromatography on silica gel using the Biotagepurification system (80 g silicycle column; solvent gradient:dichloromethane 3CV, ramp to 6:94 (2M NH₃ in methanol):CH₂Cl₂ over 15CV;254 nm monitoring wavelength; flow=45 mL/minute), to give an orangesolid (0.937 g, 59.8%). ¹H-NMR (DMSO-d₆) δ 7.85-7.81 (m, 2H), 6.82 (d,J=9.0 Hz, 1H), 3.82-3.79 (m, 2H), 3.58 (t, J=6.9 Hz, 2H), 3.08-3.04 (m,2H), 2.50-2.45 (m, 2H), 2.44-2.34 (m, 4H), 1.49-1.46 (m, 4H), 1.40-1.37(m, 2H); ESI-MS (m/z, %): 308 (MH⁺, 100).

4-(2-(Piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

A mixture of7-nitro-4-(2-(piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine(0.9 g, 2.93 mmol) in methanol (10 mL) under an argon atmosphere wastreated first with hydrazine hydrate (1.424 mL, 29.3 mmol) and then withRaney-Nickel (˜0.172 g, 2.93 mmol). The reaction was transferred to oilbath pre-heated to 65° C. After one hour, the reaction mixture waspoured over a pad of Celite. The pad of Celite was then rinsed withmethanol (25 mL). The purple filtrate was concentrated and subjected toa short column on silica gel (1:9 (2M NH₃ in MeOH):CH₂Cl₂). Thefractions were concentrated, and the residue was carried on to the nextstep. ¹H-NMR (DMSO-d₆) δ 6.49 (d, J=8.7 Hz, 1H), 6.26-6.23 (m, 2H), 4.41(s, 2H), 3.38-3.35 (m, 2H), 3.20 (t, J=7.2 Hz, 2H), 2.98-2.94 (m, 2H),2.40-2.34 (m, 6H), 1.50-1.46 (m, 4H), 1.38-1.36 (m, 2H); ESI-MS (m/z,%): 278 (MH⁺, 100).

N-(4-(2-(Piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A suspension of4-(2-(piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.90 g, 3.24 mmol) and methyl thiophene-2-carbimidothioate hydroiodide(1.85 g, 6.49 mmol) was stirred at room temperature overnight (16 hours)under an argon atmosphere. The reaction was concentrated, and theresidue was partitioned between saturated sodium bicarbonate solution(50 mL) and dichloromethane (25 mL). After stirring for an hour, themixture was poured into a separatory funnel. The organic layer wasremoved, and the aqueous layer was extracted into dichloromethane (2×20mL). The combined organic layers were dried (Na₂SO₄), filtered, andconcentrated. The residue was subjected to column chromatography onsilica gel (2:98 to 5:95 MeOH:CH₂Cl₂ followed by 5:95 (2M NH₃ inMeOH):CH₂Cl₂) to give compound 14 as yellow solid (0.288 g, 23%). ¹H-NMR(DMSO-d₆) δ 7.69 (d, J=3.3 Hz, 1H), 7.57 (d, J=4.5 Hz, 1H), 7.09-7.06(m, 1H), 6.67 (d, J=8.7 Hz, 1H), 6.52-6.47 (m, 2H), 6.39 (brs, 2H),3.56-3.53 (m, 2H), 3.39-3.33 (m, 2H), 3.03-3.01 (m, 2H), 2.45-2.39 (m,6H), 1.49-1.48 (m, 4H), 1.39-1.38 (m, 2H); ESI-MS (m/z, %): 387 (MH⁺,77), 276 (46), 194 (100).

N-(4-(2-(Piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(2-(piperidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.288 g, 0.745 mmol) in anhydrous methanol (8 mL) was treated withhydrochloric acid (1M in ether; 3.72 mL, 3.72 mmol). The reaction wasstirred at room temperature for 0.5 hours and then concentrated to givean orange solid (0.34 g, 99%). ¹H-NMR (DMSO-d₆) δ 11.21 (brs, 1H), 11.16(brs, 1H), 9.67 (brs, 1H), 8.69 (brs, 1H), 8.15 (brd, J=4.8 Hz, 1H),8.11 (brd, J=3.0 Hz, 1H), 7.38-7.35 (m, 1H), 7.11-6.98 (m, 3H),3.89-3.84 (m, 2H), 3.68-3.64 (m, 2H), 3.45-3.42 (m, 2H), 3.22-3.15 (m,2H), 3.12-3.09 (m, 2H), 2.89-2.88 (m, 2H), 1.87-1.71 (m, 5H), 1.42-1.38(m, 1H); ESI-MS (m/z, %): 387 (MH⁺, free base, 100), 194 (23); ESI-HRMScalculated for C₂₀H₂₇N₄S₂ (MH⁺, free base): 387.1671, observed:337.1657; HPLC purity: 99% by area.

Example 15 Synthesis ofN-(4-(2-(Diethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(15)

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the procedure reported in Example 11.

N,N-Diethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

A mixture of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (1 g, 5.10mmol), 2-chloro-N,N-diethylethanamine hydrochloride (1.754 g, 10.19mmol), and nBu₄NBr (0.082 g, 0.255 mmol) in CH₂Cl₂ (10 mL) and water (10mL) was stirred at room temperature for 16 hours. The mixture wasdiluted with CH₂Cl₂ (5 mL) and water (20 mL). The mixture wastransferred to a separatory funnel, and the layers were separated. Theaqueous layer was extracted with CH₂Cl₂ (2×10 mL). The combined organiclayers were dried (Na₂SO₄), filtered, and concentrated. The red crudematerial was subjected to flash chromatography on silica gel (CH₂Cl₂then 1.75-3.5% (2M NH₃ in MeOH):CH₂Cl₂) to give a brown oil (1.15 g,77%). ¹H NMR (DMSO-d₆) δ 7.86-7.81 (m, 2H), 6.81 (d, J=9 Hz, 1H),3.85-3.82 (m, 2H), 3.53 (t, J=6.6 Hz, 2H), 3.07-3.04 (m, 2H), 2.58 (t,J=6.3 Hz, 2H), 2.52-2.45 (m, 4H), 0.92 (t, J=7.2 Hz, 6H); ESI-MS (m/z,%): 298, 296 (MH⁺, 100).

4-(2-(Diethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

To a stirred solution ofN,N-diethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine(1.109 g, 3.76 mmol) in MeOH (30 mL) was added Raney-Nickel (slurry inwater; ˜0.1 g, 3.76 mmol) followed by hydrazine hydrate (1.828 mL, 37.6mmol). The mixture was immersed in a preheated oil bath and refluxed for30 minutes. The solution was cooled to room temperature, filteredthrough Celite, and washed with MeOH. The crude material was filteredthrough a silica plug (3.5% (2M NH₃ in MeOH):CH₂Cl₂). The solvent wasevaporated to give the product as dark brown oil (1.07 g, quantitative).¹H NMR (CDCl₃) δ 6.58 (d, J=8.7 Hz, 1H), 6.45 (d, J=2.7 Hz, 1H), 6.40(dd, J=2.7, 8.7 Hz, 1H), 3.53-3.50 (m, 2H), 3.30 (m, 4H), 3.03-2.99 (m,2H), 1.03 (t, J=7.2 Hz, 6H); ESI-MS (m/z, %): 268, 266 (MH⁺, 100), 100(52).

N-(4-(2-(Diethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

Methyl thiophene-2-carbimidothioate hydroiodide (2.266 g, 7.94 mmol) wasadded to a mixture of4-(2-(diethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(1.054 g, 3.97 mmol) in EtOH (20 mL). The mixture was stirred for 2 daysat room temperature. The reaction was quenched with saturated sodiumbicarbonate solution (30 mL) and extracted with CH₂Cl₂ (50 mL). Theaqueous phase was washed with CH₂Cl₂ (50 mL). The combined organicfractions were washed with brine (50 mL) and dried (Na₂SO₄). The crudematerial was subject to flash chromatography on silica gel (CH₂Cl₂followed by 3.5% (2M NH₃ in MeOH):CH₂Cl₂). The collected fractions wereconcentrated, and the sample was subjected again to flash chromatographyon silica gel (2.5% MeOH:CH₂Cl₂). The collected fractions wereconcentrated to give compound 15 as a brown solid. (1.23 g, 83%). ¹H NMR(CDCl₃) δ 7.40-7.36 (m, 2H), 7.05 (dd, J=3.9, 4.8 Hz, 1H), 6.73 (m, 3H),3.65-3.61 (m, 1H), 3.39 (t, J=7.2 Hz, 2H), 3.05-3.02 (m, 2H), 2.67-2.54(m, 6H), 1.04 (t, J=7.2 Hz, 6H); ESI-MS (m/z, %): 377, 375 (MH⁺, 35),278, 276 (100).

N-(4-(2-(Diethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

To a stirred solution ofN-(4-(2-(diethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(1.149 g, 3.07 mmol) in MeOH (5 mL) was added 1M HCl in ether (15.35 mL,15.35 mmol) at room temperature. The mixture was stirred for 5 minutesunder argon atmosphere and concentrated to give a yellow solid (1.37 g,quantitative). ¹H NMR (DMSO-d₆) δ 11.24 (s, 2H), 9.68 (s, 1H), 8.69 (s,1H), 8.15-8.13 (m, 2H), 7.37-7.34 (m, 1H), 7.12-7.00 (m, 3H), 3.88-3.83(m, 2H), 3.69-3.66 (m, 2H), 3.17-3.10 (m, 8H), 1.25 (t, J=7.2 Hz, 6H);ESI-MS (m/z, %): 377, 375 (MH⁺, free base, 58), 278, 276 (100); ESI-HRMScalculated for C₁₉H₂₅N₄S₂ (MH⁺, free base), calculated: 375.1671,observed: 375.1667; HPLC: 95% by area.

Example 16 Synthesis ofN-(4-(2-(2-hydroxyethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride (16)

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the procedure reported in Example 11.

2-Chloro-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

2-Chloroacetyl chloride (4.08 mL, 50.9 mmol) was added dropwise to asolution containing 7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (5 g,25.5 mmol) in toluene (30 mL). The mixture was refluxed at 110° C. for20 minutes. The mixture was quenched with saturated sodium bicarbonatesolution (100 mL). The mixture was transferred to a separatory funneland extracted with EtOAc (4×100 mL). The crude material was filteredthrough a pad of silica gel and washed with EtOAc. The filtrate wasconcentrated to give a yellow-brown solid. ¹H-NMR (DMSO-d₆) δ 8.12 (d,J=2.7 Hz, 1H), 7.94 (dd, J=2.7, 9.0 Hz, 1H), 7.72 (d, J=9.0 Hz, 1H),4.63 (s, 2H), 3.96 (t, J=5.1 Hz, 2H), 3.32 (t, J=5.4 Hz, 2H, overlapwith solvent peak); ESI-MS (m/z, %): 273 (MH⁺, 100), 295 (40).

2-(2-Hydroxyethylamino)-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

A solution of2-chloro-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone (1.0 g,3.67 mmol) in THF (20 mL) was treated with 4M HCl in 1,4-dioxane (4.58mL, 18.33 mmol), followed by the slow addition of a solution of2-aminoethanol (2.207 mL, 36.7 mmol) in water (4 mL). The dark solutionwas stirred at room temperature for 24 hours. The mixture was dilutedwith EtOAc (50 mL) and saturated sodium bicarbonate (100 mL), thenstirred for 20 minutes. The contents were poured into a separatoryfunnel, and the organic layer was separated, dried (Na₂SO₄), filtered,and concentrated to give a dark yellow residue. This residue wassubjected to flash chromatography on silica gel using 5:95 MeOH:CH₂Cl₂followed by 5:95 (2M NH₃ in MeOH):CH₂Cl₂ to give a yellow residue (320mg, 29.4%). ¹H-NMR (CDCl₃) δ 8.14 (d, J=2.7 Hz, 1H), 7.95 (dd, J=2.7,9.0 Hz, 1H), 7.37 (d, J=9.0 Hz, 1H), 4.02 (t, J=5.1 Hz, 2H), 3.61 (t,J=5.1 Hz, 2H), 3.57 (s, 2H), 3.28 (t, J=5.4 Hz, 2H), 2.77 (t, J=4.8 Hz,2H); ESI-MS (m/z, %): 298 (MH⁺, 100%).

tert-Butyl2-hydroxyethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate

A solution of2-(2-hydroxyethylamino)-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone(300 mg, 1.009 mmol) in THF (15 mL) was treated with Boc anhydride (231mg, 1.059 mmol) followed by triethylamine (0.422 mL, 3.03 mmol). Theyellow solution was stirred at room temperature for 18 hours(overnight). The solution was diluted with EtOAc (20 mL) and treatedwith saturated sodium bicarbonate solution (20 mL). The mixture wastransferred to a separatory funnel and extracted. After extraction, theorganic layer was separated, dried (Na₂SO₄), and filtered through asilica gel plug. The pad was rinsed with EtOAc (10 mL), and the filtratewas concentrated to give a yellow residue. This residue was subjected tosilica gel chromatography using the Biotage purification system (column:Silicycle 40 g; 30% EtOAc/70% hexanes gradient to 90% EtOAc/10% hexanesover 10 column volumes; flow rate=35 mL/min; collection wavelength: 254nm). A yellow residue was obtained after drying under reduced pressure(270 mg, 67.3%). ¹H-NMR (CDCl₃) (mixture of rotamers) δ 8.15 (dd, J=2.7,8.1 Hz, 1H), 7.94 (dd, 1=2.4, 8.7 Hz, 1H), 7.58 and 7.31 (2×d, J=9.0 Hz,1H), 4.41 (brs, 1H), 4.12-3.88 (m, 4H), 3.82-3.66 (m, 2H), 3.48 and 3.11(2×t, J=4.5 Hz, 2H), 3.29 (t, J=4.5 Hz, 2H), 1.46 and 1.40 (2×s, 9H);ESI-MS (m/z, %): 420 (M+Na, 70), 398 (MH⁺, 40), 298 (100).

tert-Butyl2-hydroxyethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

A solution of tert-butyl2-hydroxyethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate(250 mg, 0.629 mmol) in THF (10 mL) was cooled to 0° C. then treatedwith borane in THF (1M) (6.29 mL, 6.29 mmol). The resulting brightyellow solution was allowed to warm to room temperature and then stirredat this temperature for 2.5 days. The mixture was cooled to 0° C., andthe reaction was then carefully quenched with MeOH (dropwise addition atfirst; 20 mL total). The cooling bath was removed, and the yellowsolution was stirred for 20 minutes then concentrated. The orangeresidue was dissolved in MeOH (50 mL) and concentrated to dryness. Thisresidue was dissolved in EtOAc (20 mL) and filtered through a pad ofsilica gel. The silica pad was eluted with EtOAc (100 mL). The filtratewas concentrated to give an orange residue which was dried under reducedpressure for 4 hours (230 mg, 95%). ¹H-NMR (CDCl₃) δ 7.97 (brs, 1H),7.87 (brd, J=8.7 Hz, 1H), 6.85-6.71 (m, 1H), 3.81 (brs, 5H), 3.61 (brs,2H), 4.54-3.31 (m, 4H), 3.02 (overlapping t, 2H), 1.47 (brs, 9H); ESI-MS(m/z, %): 406 (M+Na, 60), 384 (MH⁺, 10), 328 (60), 284 (100).

tert-Butyl2-hydroxyethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

A round bottom flask containing tert-butyl2-hydroxyethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(220 mg, 0.574 mmol) under argon was charged with palladium on activatedcarbon (10% wt; 61.0 mg, 0.057 mmol). The mixture was purged with argonprior to the addition of EtOH (20 mL). The resulting suspension wasevacuated using a pump, and hydrogen was let into the system via aballoon. The mixture was stirred under a balloon filled with hydrogenfor 3 hours. The hydrogen balloon was removed, and methylthiophene-2-carbimidothioate hydroiodide (327 mg, 1.147 mmol) was addedto the reaction. The suspension was stirred at room temperature for 17hours (overnight). After this time, the mixture was filtered through apad of Celite, which was then rinsed with MeOH (10 mL). The filtrate wasconcentrated, and the residue was partitioned between saturated sodiumbicarbonate solution (50 mL) and CH₂Cl₂ (100 mL). The mixture wastransferred to a separatory funnel and extracted. After extraction, theorganic layer was separated, dried (Na₂SO₄), filtered, and concentratedto give a dark residue. This residue was subjected to flashchromatography on silica gel using 2:98 MeOH/CH₂Cl₂ followed by 3.5:96.5(2M NH₃ in MeOH):CH₂Cl₂ to give a yellow solid (140 mg, 52.7%). ¹H-NMR(DMSO-d₆) δ 7.69 (brd, J=3.0 Hz, 1H), 7.57 (d, J=−5.1 Hz, 1H), 7.07 (dd,J=3.9, 4.8 Hz, 1H), 6.82-6.75 (m, 1H), 6.53-6.42 (m, 2H), 6.32 (brs,2H), 4.73 (t, J=5.4 Hz, 1H), 3.54 (brs, 2H), 3.50-3.42 (m, 2H), 3.38(brs, 4H, overlap with solvent peak), 3.27-3.19 (m, 2H), 3.08-2.96 (m,2H), 1.41 (brs, 9H); ESI-MS (m/z, %): 463 (MH⁺, 100), 398 (60); HPLCpurity: 96.4% a/a.

N-(4-(2-(2-Hydroxyethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

To a solution of tert-butyl2-hydroxyethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(130 mg, 0.281 mmol) in MeOH (10 mL) was added 3N HCl (2.81 mL, 8.43mmol). The yellow solution was heated at 75° C. for 30 minutes. At thistime, the reaction was filtered and concentrated to half its volume. Theyellow solution was then extracted with 2×50 mL CH₂Cl₂, and the aqueouslayer was concentrated to dryness to give a yellow residue. This residuewas dried under reduced (high vacuum) pressure to give compound 16 asyellow powder. ¹H-NMR (CD₃OD) δ 7.98-7.94 (m, 2H), 7.29 (pseudo t, J=4.2Hz, 1H), 7.04-6.94 (m, 3H), 3.79 (brt, J=4.8 Hz, 2H), 3.72-3.65 (m, 4H),3.28 (brs, 2H), 3.16 (brt, J=5.1 Hz, 2H), 3.09-3.06 (m, 2H); ESI-MS(m/z, %): 363 (MH⁺, free base, 100), 276 (90); ESI-HRMS calculated forC₁₇H₂₃N₄O₁S₂ (MH⁺, free base): 363.1307, observed: 363.1316; HPLCpurity: 96.2% a/a.

Example 17 Synthesis of2-(methyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)amino)aceticacid dihydrochloride (17)

N,N-Dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

Prepared according to the procedure reported in Example 8.

N-Methyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

A solution ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine(1.65 g, 6.17 mmol) in ClCH₂CH₂Cl (20 mL) was cooled to 0° C. (ice/waterbath) then treated with alpha chloroethylchoroformate (1.009 mL, 9.26mmol) dropwise. The resulting suspension was heated at 88° C. for 1.5hours then concentrated to dryness. The residue was dissolved in MeOH(50 mL) and heated at reflux for 2 hours. The dark mixture wasconcentrated and partitioned between saturated sodium bicarbonatesolution (50 mL) and CH₂Cl₂ (200 mL). The mixture was transferred to aseparatory funnel and extracted. After extraction, the organic layer wasseparated, dried (Na₂SO₄), filtered, and concentrated to give a brownresidue. This residue was subjected to flash chromatography on silicagel using 5:95 (2M NH₃ in MeOH):CH₂Cl₂ as the eluent to give a yellowresidue (980 mg, 62.7%). ¹H-NMR (CDCl₃) δ 7.97 (d, J=2.7 Hz, 1H), 7.87(d, J=2.7, 9.3 Hz, 1H), 6.68 (d, J=9.3 Hz, 1H), 3.86-3.82 (m, 2H), 3.55(t, J=6.6 Hz, 2H), 3.04-3.01 (m, 2H), 2.87 (t, J=6.6 Hz, 2H), 2.49 (s,3H), 1.32 (brs, 1H).

tert-Butyl2-(methyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)amino)acetate

A solution ofN-methyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine (0.95 g,3.75 mmol), diisopropylethyl amine (1.960 mL, 11.25 mmol), andtert-butyl bromoacetate (0.61 mL, 4.13 mmol) in DMF (20 mL) was heatedat 55° C. for 17 hours (overnight). The yellow solution was diluted withbrine (40 mL) and extracted with EtOAc (100 mL). The organic extract wasseparated and rinsed with brine (20 mL). The organic layer was dried(Na₂SO₄), filtered, and concentrated to give a yellow residue. Thisresidue was subjected to flash chromatography on silica gel using2.5:97.5 MeOH:CH₂Cl₂ to give a yellow brown residue. Two fractions werecollected, with the latter containing a non-polar yellow spot (950 mg,68.9%). ¹H-NMR (CDCl₃) δ 7.96 (d, J=2.7 Hz, 1H), 7.88 (d, J=2.7, 9.3 Hz,1H), 6.65 (d, J=9.3 Hz, 1H), 3.88-3.83 (m, 2H), 3.55 (t, J=7.2 Hz, 2H),3.20 (s, 2H), 3.02-2.99 (m, 2H), 2.76 (t, J=7.2 Hz, 2H), 2.44 (s, 3H),1.46 (s, 9H); ESI-MS (m/z, %): 368 (MH⁺, 20), 312 (100).

tert-Butyl2-(2-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)(methyl)amino)acetate

A round bottom flask containing tert-butyl2-(methyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)amino)acetate(925 mg, 2.52 mmol) was purged with argon and then charged withpalladium on carbon (10% wt; 268 mg, 0.252 mmol). To this mixture wasadded EtOH (40 mL). The resulting suspension was evacuated using a pump,and hydrogen was let into the system via a balloon. The mixture wasstirred under a balloon filled with hydrogen for 3 hours. The balloonwas removed, and argon was bubbled through the suspension for 10minutes. The mixture was filtered through a pad of Celite, and theCelite pad was rinsed with methanol (20 mL). The filtrate wasconcentrated, and the residue was dried under reduced pressure to givethe title compound (850 mg, quantitative). ¹H-NMR (CDCl₃) δ 6.59 (d,J=8.7 Hz, 1H), 6.45 (d, J=2.4 Hz, 1H), 6.40 (dd, J=2.7, 8.4 Hz, 1H),3.54-3.45 (m, 2H), 3.33 (t, J=7.2 Hz, 2H), 3.27 (brs, 2H), 3.20 (s, 2H),3.04-2.98 (m, 2H), 2.71 (t, J=7.5 Hz, 2H), 2.42 (s, 3H), 1.46 (s, 9H);ESI-MS (m/z, %): 338 (MH⁺, 95), 282 (100).

tert-Butyl2-(methyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)amino)acetate

To a solution of tert-butyl2-((2-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)(methyl)amino)acetate(850 mg, 2.52 mmol) in EtOH (25 mL) was added methylthiophene-2-carbimidothioate hydroiodide (1.43 g, 5.04 mmol). Theresulting suspension was stirred at room temperature for 17 hours(overnight). At this time, the suspension was diluted with 20 mL CH₂Cl₂to give a dark yellow solution. Argon was then bubbled through thissolution for 30 minutes. The solution was transferred to a separatoryfunnel containing saturated sodium bicarbonate solution (50 mL) andCH₂Cl₂ (100 mL) then extracted. After extraction, the organic layer wasseparated, dried (Na₂SO₄), filtered, and concentrated to give a darkresidue. This residue was subjected to flash chromatography on silicagel using 2:98 MeOH:CH₂Cl₂ followed by 2.5:97.5 (2M NH₃ in MeOH):CH₂Cl₂to give a yellow-green residue (420 mg, 37.3%). Some impure fractionswere collected (˜340 mg). ¹H-NMR (CDCl₃) δ 7.43-7.35 (m, 2H), 7.06(pseudo t, J=4.8 Hz, 1H), 7.71 (brd, J=8.1 Hz, 2H), 6.65 (dd, J=1.8, 8.4Hz, 1H), 4.91 (brs, 2H), 3.66-3.58 (m, 2H), 3.43 (t, J=7.2 Hz, 2H), 3.21(s, 2H), 3.07-2.99 (m, 2H), 2.74 (t, J=7.5 Hz, 2H), 2.45 (s, 3H), 1.47(s, 9H); ESI-MS (m/z, %): 447 (MH⁺, 100), 391 (50); HPLC purity: 99.7%a/a.

2-(Methyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)amino)aceticacid dihydrochloride

An oven dried round bottom flask, equipped with a magnetic stirbar underargon, was charged with tert-butyl2-(methyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)amino)acetate(200 mg, 0.448 mmol) in CH₂Cl₂ (3 mL), and the solution was cooled to 0°C. (ice/water bath). To this solution was added anisole (0.098 mL, 0.896mmol) followed by the dropwise addition of trifluoroacetic acid (2.92mL, 26.9 mmol). The reaction was stirred at 0° C. for 20 minutes. Theice bath was then removed, and the mixture was stirred for 3 additionalhours. At this time, the mixture was concentrated and treated with 4.0 MHCl in 1,4-dioxane (4 mL). The resulting suspension was diluted withEt₂O (20 mL) and then stirred for 20 minutes. The mixture was filtered,and the solid was washed with Et₂O (20 mL). The solid was dried underreduced pressure to give compound 17 as light yellow powder (200 mg,96%). An HPLC analysis indicated that the sample contained ˜3.4% of thestarting material. ¹H-NMR (CD₃OD) δ 7.99 (d, J=5.1 Hz, 1H), 7.97 (d,J=3.6 Hz, 1H), 7.31 (pseudo t, J=4.5 Hz, 1H), 7.11-6.99 (m, 2H), 6.95(d, J=8.7 Hz, 1H), 4.16 (s, 2H), 3.81 (brt, J=6.9 Hz, 2H), 3.68 (brt,J=4.8 Hz, 2H), 3.52-3.39 (m, 2H), 3.10 (brt, J=4.8 Hz, 2H), 3.03 (s,3H); ESI-MS (positive ion mode): 391 (MH⁺, free base, 100); ESI-MS(negative ion mode): 389 (M−1, free base, 100); ESI-HRMS calculated forC₁₈H₂₃N₄O₂S₂ (MH⁺, free base): 391.1256, Observed: 391.1255; HPLCpurity: 96.64% a/a.

Example 18 Synthesis of(S)-1-(2-(7-(Thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)pyrrolidine-2-carboxylicacid dihydrochloride (18)

2-Chloro-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

Prepared according to the reported procedure in Example 16.

(S)-tert-Butyl-1-(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)pyrrolidine-2-carboxylate

A solution of2-chloro-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone (1.0 g,3.67 mmol) in THF (20 mL) was treated with (S)-tert-butylpyrrolidine-2-carboxylate (0.942 g, 5.50 mmol), followed by saturatedsodium bicarbonate (4 mL). The mixture was heated at 55° C. for 3 hoursthen stirred at room temperature overnight (17 hours). The mixture wasdiluted with EtOAc (50 mL) and saturated sodium bicarbonate (10 mL) thenstirred for 20 minutes. The contents were poured into a separatoryfunnel, and the organic layer was separated, dried (Na₂SO₄), filtered,and concentrated to give a dark yellow residue. This residue wassubjected to silica gel chromatography using the Biotage purificationsystem (column: Silicycle 40 g; 20:80 EtOAc:hexanes gradient to 60:40EtOAc:hexanes over 8 column volumes; flow rate: 35 mL/min; collectionwavelength: 254 nm). A yellow residue was obtained (1.47 g, 98%). ¹H-NMR(CDCl₃) δ 8.09 (d, J=2.4 Hz, 1H), 7.91 (dd, J=2.4, 8.7 Hz, 1H), 7.75 (d,J=9.0 Hz, 1H), 4.10 (brs, 2H), 3.74 (brd, J=13.2 Hz, 1H), 3.40 (brd,J=13.5 Hz, 1H), 3.37-3.21 (m, 3H), 3.12-3.04 (m, 1H), 2.61 (brdd, J=8.1,16.2 Hz, 1H), 2.21-2.07 (m, 1H), 1.95-1.77 (m, 3H), 1.43 (s, 9H); ESI-MS(m/z, %): 408 (MH⁺, 50), 352 (100).

(S)-tert-Butyl-1-(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)pyrrolidine-2-carboxylate

1M Borane in THF (17.18 mL, 17.18 mmol) was added to (S)-tert-butyl1-(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)pyrrolidine-2-carboxylate(1.4 g, 3.44 mmol), and the resulting yellow solution was stirred atroom temperature overnight (17 hours). The yellow solution was cooled to0° C. and carefully quenched with MeOH (10 mL). The solution wasconcentrated, dissolved in MeOH (50 mL), and concentrated again to givea dark yellow residue. This residue was subjected to silica gelchromatography using the Biotage purification system (column: Silicycle80 g; 20:80 EtOAc:hexanes gradient to 60:40 EtOAc:hexanes over 10 columnvolumes; flow rate=45 mL/min; collection wavelength: 254 nm). A yellowresidue was obtained (550 mg, 40.7%). ¹H-NMR (CDCl₃) δ 7.96 (d, J=2.7Hz, 1H), 7.87 (dd, J=2.7, 9.3 Hz, 1H), 6.68 (d, J=9.3 Hz, 1H), 3.86-3.82(m, 2H), 3.57 (t, J=7.5 Hz, 2H), 3.24-3.10 (m, 2H), 3.05-2.96 (m, 3H),2.75-2.63 (m, 1H), 2.52-2.41 (m, 1H), 2.15-2.04 (m, 1H), 1.97-1.78 (m,3H), 1.44 (s, 9H); ESI-MS (m/z, %): 394 (MH⁺, 25), 338 (100).

(S)-tert-Butyl-1-(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)pyrrolidine-2-carboxylate

A round bottom flask containing (S)-tert-butyl1-(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)pyrrolidine-2-carboxylate(525 mg, 1.334 mmol) was purged with argon. This flask was charged withpalladium on activated carbon (10% wt; 142 mg, 0.133 mmol) followed byEtOH (25 mL). The resulting suspension was evacuated using a pump, andhydrogen was let into the system via a balloon. The mixture was stirredunder a balloon filled with hydrogen for 2 hours. At this time, thehydrogen balloon was removed, and methyl thiophene-2-carbimidothioatehydroiodide (761 mg, 2.67 mmol) was added to the mixture. The suspensionwas stirred at room temperature for 17 hours (overnight). At this time,the mixture was filtered through a pad of Celite, and the pad was rinsedwith MeOH (20 mL). The filtrate was concentrated, and the residuepartitioned between saturated sodium bicarbonate solution (50 mL) andCH₂Cl₂ (100 mL). The mixture was transferred to a separatory funnel andextracted. After extraction, the organic layer was separated, dried(Na₂SO₄), filtered, and concentrated to give a dark residue. Thisresidue was subjected to flash chromatography on silica gel using 2:98MeOH:CH₂Cl₂ followed by 3.5:96.5 (2M NH₃ in MeOH):CH₂Cl₂ to give ayellow-green residue (398 mg, 63.1%). ¹H-NMR (CDCl₃) δ 7.39 (dd, J=1.2,5.1 Hz, 1H), 7.37 (dd, J=1.2, 3.9 Hz, 1H), 7.05 (dd, J=3.6, 4.8 Hz, 1H),6.73 (brd, J=5.1 Hz, 1H), 6.71 (brs, 1H), 6.65 (dd, J=2.1, 8.7 Hz, 1H),4.86 (brs, 2H), 3.63-3.60 (m, 2H), 3.44 (t, J=7.5 Hz, 2H), 3.25-3.19 (m,1H), 3.11 (brdd, J=5.4, 8.4 Hz, 1H), 3.04-2.94 (m, 3H), 2.71-2.59 (m,1H), 2.45 (brdd, J=7.8, 16.2 Hz, 1H), 2.13-1.99 (m, 1H), 1.97-1.74 (m,3H), 1.45 (s, 9H); ESI-MS (m/z, %): 473 (MH⁺, 90), 417 (85), 276 (100);HPLC Purity: 99.6% a/a.

(S)-1-(2-(7-(Thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)pyrrolidine-2-carboxylicacid dihydrochloride

A solution of (S)-tert-butyl1-(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)pyrrolidine-2-carboxylate(200 mg, 0.423 mmol) in CH₂Cl₂ (5 mL) was treated with anisole (0.092mL, 0.846 mmol). The reaction was cooled to 0° C., and trifluoroaceticacid (2.76 mL, 25.4 mmol) was then added. The mixture was stirred at 0°C. for 2 hours then concentrated to dryness. To this residue was added4M HCl in dioxane (3.17 mL, 12.69 mmol), and the resulting suspensionwas stirred for 20 minutes. This mixture was diluted with Et₂O (10 mL)and stirred for 1 hour. The solid was filtered, washed with Et₂O (10mL), and then dried under reduced pressure. The solid was dissolved in3N HCl (4.0 mL, 12.00 mmol) and heated at 50° C. for 1 hour. The yellowsolution was concentrated and dried under reduced pressure to givecompound 18 as yellow solid (200 mg, 97%). ¹H-NMR (CD₃OD) δ 7.98-7.94(m, 2H), 7.28 (dd, J=4.2, 4.8 Hz, 1H), 7.05-7.01 (m, 2H), 6.92 (d, J=8.7Hz, 1H), 4.42 (dd, J=7.8, 9.0 Hz, 1H), 3.85-3.56 (m, 6H), 3.48-3.29 (m,2H), 3.13-3.03 (m, 2H), 2.62-2.47 (m, 1H), 2.26-2.10 (m, 2H), 2.08-1.92(m, 1H); ESI-MS (m/z, %): 417 (MH⁺, free base, 100); ESI-HRMS calculatedfor C₂₀H₂₅N₄O₂S₂ (MH⁺, free base): 417.1419, observed: 417.1399; HPLCpurity: 99.5% a/a; Optical Rotation: ²⁵[α]₅₈₉=−27.0°, c=0.52 in MeOH.

Example 19 Synthesis ofN-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamide(19)

N,N-Dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

Prepared according to the procedure reported in Example 8.

4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

To a solution ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine(0.9695 g, 3.63 mmol) in MeOH (20 mL) was added Raney-Nickel (slurry inwater; ˜0.1 g, 3.63 mmol) followed by hydrazine hydrate (1.82 mL, 37.4mmol). The mixture was immersed in a preheated oil bath and refluxed for20 minutes. The solution was cooled to room temperature and filteredthrough a pad of Celite. The filter pad was washed with MeOH, and thecrude material was concentrated. The crude material was filtered througha plug of silica gel (2.5:97.5 (2M NH₃ in MeOH):CH₂Cl₂). The collectedfractions gave brown viscous oil (0.84 g, 99%). ¹H NMR (CDCl₃) δ 6.58(d, J=8.7 Hz, 1H), 6.45 (d, J=2.7 Hz, 1H), 6.40 (dd, J=2.7, 8.7 Hz, 1H),3.51-3.48 (m, 2H), 3.30 (t, J=7.2 Hz, 2H), 3.03-3.00 (m, 2H), 2.48 (t,J=7.5 Hz, 2H), 2.27 (s, 6H); ESI-MS (m/z, %): 240, 238 (MH⁺, 100).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamide

Benzyl furan-2-carbimidothioate hydrobromide (1.139 g, 3.82 mmol) wasadded to a mixture of4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.829 g, 3.49 mmol) in EtOH (20 mL). The mixture was stirred for 2 daysunder argon atmosphere. The solution was quenched with saturated sodiumbicarbonate (50 mL). The solution was transferred to a separatoryfunnel, diluted with water (50 mL), and extracted with CH₂Cl₂ (50 mL).The aqueous phase was washed with CH₂Cl₂ (50 mL). The combined organicfractions were washed with brine (50 mL) and dried (Na₂SO₄). The crudematerial was subjected to flash chromatography on silica gel (2.5-5% (2MNH₃ in MeOH):CH₂Cl₂). The collected fractions gave compound 19 as abrown oil (1.03 g, 90%). ¹H NMR (CDCl₃) δ 7.46 (s, 1H), 7.03 (brs, 1H),6.73-6.68 (m, 3H), 6.50 (brs, 1H), 5.03 (brs, 2H), 3.62-3.59 (m, 2H),3.40 (t, J=7.2 Hz, 2H), 3.05-3.02 (m, 2H), 2.52 (t, J=7.5 Hz, 2H), 2.29(s, 6H); ESI-MS (m/z, %): 333, 331 (MH⁺, 91), 260, 262 (100).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamidedihydrochloride

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamide(0.166 g, 0.504 mmol) was dissolved in MeOH (2 mL). 1M HCl in ether(2.52 mL, 2.52 mmol) was added to the solution at room temperature, andthe reaction was stirred for 5 minutes under argon atmosphere. Themixture was concentrated to give a light yellow solid (0.18 g, 93%). ¹HNMR (DMSO-d₆) δ 11.36 (s, 1H), 11.25 (brs, 1H), 9.68 (s, 1H), 8.69 (s,1H), 8.23 (s, 1H), 7.91 (d, J=2.7 Hz, 1H), 7.07-6.96 (m, 3H), 6.90 (d,J=1.8 Hz, 1H), 3.82-3.77 (m, 2H), 3.67-3.63 (m, 2H), 3.25-3.22 (m, 2H),3.12-3.09 (m, 2H), 2.80 (s, 3H), 2.78 (s, 3H); ESI-MS (m/z, %): 333, 331(MH⁺, free base, 100), 260, 262 (57); HRMS calculated for C₁₇H₂₃N₄OS(MH⁺, free base): calculated: 331.1587, observed: 331.1589; HPLC purity:98% by area.

Example 20 Synthesis ofN-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamide(20)

N,N-Dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

Prepared according to the procedure reported in Example 8.

N-Methyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine:1-Chloroethyl chloroformate (0.612 mL, 5.61 mmol) was added to asolution ofN,N-dimethyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine (1.0g, 3.74 mmol) in 1,2-dichloroethane (20 mL) at 0° C. in an argonatmosphere. The solution was brought to room temperature and refluxedunder vigorous stirring for 3 hours. The solution was concentrated andthen refluxed in MeOH (20 mL). The solution was concentrated to givedark brown viscous oil (1.05 g, quantitative). ¹H NMR (CDCl₃) δ 7.96 (d,J=2.4 Hz, 1H), 7.86 (dd, J=2.7, 9.3 Hz, 1H), 6.67 (d, J=9.3 Hz, 1H),3.85-3.82 (m, 2H), 3.55 (t, J=6.9 Hz, 2H), 3.03-3.00 (m, 2H), 2.86 (t,J=6.9 Hz, 2H), 2.48 (s, 3H); ESI-MS (m/z, %): 256, 254 (MH⁺, 100).

tert-Butylmethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution ofN-methyl-2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine (1.022g, 4.04 mmol) and triethylamine (1.135 mL, 8.08 mmol) in dioxane (20 mL)under argon atmosphere was added di-tert-butyl dicarbonate (0.984 mL,4.24 mmol). The resulting solution was stirred overnight at roomtemperature. The solution was then diluted with water (50 mL) andextracted with CH₂Cl₂ (50 mL). The organic phase was washed with brine(50 mL) and dried (Na₂SO₄). The crude material was subject to flashchromatography on silica gel (20-50% EtOAc:hexanes). The collectedfractions were concentrated to give yellow-brown oil (0.95 g, 67%). ¹HNMR (CDCl₃) δ 7.97 (s, 1H), 7.87 (d, J=9.0 Hz, 1H), 6.76-6.66 (m, 1H),3.80 (brs, 2H), 3.60-3.54 (m, 2H), 3.47-3.45 (m, 2H), 3.03-2.99 (m, 2H),2.92-2.88 (m, 3H), 1.42 (s, 9H); ESI-MS (m/z, %): 278, 276 (M+Na, 72),356, 354 (MH⁺, 19), 298, 300 (100), 256, 254 (74).

tert-Butyl2-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(methyl)carbamate

To a solution of tert-butylmethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(0.8811 g, 2.493 mmol) in MeOH (20 mL) was added Raney-Nickel (slurry inwater; ˜0.5 g, 2.493 mmol) followed by hydrazine hydrate (1.213 mL,24.93 mmol). The mixture was then immersed in a preheated oil bath andrefluxed for 5 minutes. The solution was cooled to room temperature andfiltered through a pad of Celite. The filter pad was washed with MeOH,and the crude material was concentrated. The crude material was thenfiltered through a silica plug (70% EtOAc/hexanes). The collectedfractions were concentrated to give brown oil (0.78 g, 98%). ¹H NMR(CDCl₃) δ 6.64-6.58 (m, 1H), 6.45 (d, J=2.4 Hz, 1H), 6.39 (d, J=8.4 Hz,1H), 3.53-3.50 (m, 2H), 3.39-3.28 (m, 4H), 3.01-2.98 (m, 2H), 2.90 (s,3H), 1.45 (s, 9H); ESI-MS (m/z, %): 326, 324 (MH⁺, 95), 268 (100).

tert-Butyl2-(7-(furan-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(methyl)carbamate

Benzyl furan-2-carbimidothioate hydrobromide (1.199 g, 4.02 mmol) wasadded to a mixture of tert-butyl2-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(methyl)carbamate(0.763 g, 2.361 mmol) in EtOH (20 mL). The mixture was stirred underroom temperature overnight. The mixture was quenched with saturatedsodium bicarbonate solution (50 mL), diluted with water (50 mL), andextracted with CH₂Cl₂ (2×50 mL). The organic phase was washed with brine(50 mL) and dried (Na₂SO₄). The crude material was subject to flashchromatography on silica gel (2.5-5% (2M NH₃ in MeOH):CH₂Cl₂). Thecollected fractions were concentrated to give a white foam (0.88 g,90%). ¹H NMR (CDCl₃) δ 7.85 (s, 1H), 7.44 (s, 1H), 6.74-6.69 (m, 1H),6.62 (d, J=7.5 Hz, 1H), 4.72 (brs, 2H), 3.64-3.60 (m, 2H), 3.41 (s, 4H),3.04-3.01 (m, 2H), 2.92 (s, 3H), 1.45 (s, 9H); ESI-MS (m/z, %): 419, 417(MH⁺, 100).

N-(4-(2-(Methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamide

A solution of tert-butyl2-(7-(furan-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(methyl)carbamate(0.823 g, 1.978 mmol) in CH₂Cl₂ (20 mL) was cooled to 0° C. and wastreated with trifluoroacetic acid (5 mL). The mixture was stirred at 0°C. under argon atmosphere for 2 hours. The mixture was quenched with 1NNaOH solution (70 mL) and transferred to a separatory funnel. Thesolution was diluted with water (50 mL) and extracted with CH₂Cl₂ (2×50mL). The organic phase was washed with brine (50 mL) and dried (Na₂SO₄).The crude material was concentrated and subjected to flashchromatography on silica gel (5-15% (2M NH₃MeOH):CH₂Cl₂). The collectedfractions were concentrated to give compound 20 as a yellow viscous oil(0.47 g, 76%). ¹H NMR (CDCl₃) δ 7.85 (s, 1H), 7.44-7.43 (m, 1H),6.77-6.74 (m, 2H), 6.70 (d, J=2.4 Hz, 1H), 6.62 (dd, J=2.4, 8.7 Hz, 1H),3.59-3.56 (m, 2H), 3.47 (s, 1H), 3.40 (t, J=6.6 Hz, 2H), 3.05-3.01 (m,2H), 2.81 (t, J=6.3, 2H), 2.48 (s, 3H); ESI-MS (m/z, %): 319, 317 (MH⁺,96), 260 (100).

N-(4-(2-(Methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamidedihydrochloride

N-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)furan-2-carboximidamide(0.4353 g, 1.376 mmol) was dissolved in MeOH (3 mL). 1M HCl in ether(6.88 mL, 6.88 mmol) was added to the solution at room temperature, andthe reaction was stirred for 5 minutes under argon atmosphere. Themixture was concentrated to give a yellow solid (0.59 g, quantitative).¹H NMR (DMSO-d₆) δ 11.31 (s, 1H), 9.58 (s, 1H), 9.30 (brs, 2H), 8.82 (s,1H), 8.57 (s, 1H), 7.98 (s, 1H), 7.30 (d, J=1.2 Hz, 1H), 7.06-7.03 (m,2H), 6.97 (dd, J=2.4, 8.7 Hz, 1H), 3.74-3.63 (m, 4H), 3.13-3.05 (m, 4H),2.58-2.55 (m, 3H); ESI-MS (m/z, %): 319, 317 (MH⁺, free base, 100), 260(98); HRMS calculated for C₁₆H₂₀N₄OS (MH⁺, free base), calculated:317.1430, observed: 317.1417; HPLC purity: 99% by area.

Example 21 Synthesis ofN-(4-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(21)

7-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the procedure reported in Example 11.

4-(3-Chloropropyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

A solution of 7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (1.0 g, 5.10mmol) and 1-chloro-3-iodopropane (1.075 mL, 10.19 mmol) in CH₂Cl₂ (10mL) was treated with 50% NaOH solution (10 mL), followed bytetrabutylammonium bromide (0.082 g, 0.255 mmol) at room temperature.The resulting mixture was stirred overnight (16 hours) at roomtemperature and then refluxed for 4 hours. The reaction was brought toroom temperature and diluted with water (50 mL), and the product wasextracted into CH₂Cl₂ (3×25 mL). The combined CH₂Cl₂ layers were washedwith brine (20 mL) and dried (Na₂SO₄). The solvent was evaporated, andthe crude material was purified by column chromatography (1:4 to 2:3EtOAc:hexanes) to obtain the title compound (0.65 g, 47%) as a solid. ¹HNMR (CDCl₃) δ 7.96 (d, 1H, J=2.7 Hz), 7.88 (dd, 1H, J=2.7, 9.3 Hz), 6.64(d, 1H, J=9.3 Hz), 3.84-3.80 (m, 2H), 3.65-3.60 (m, 4H), 3.05-3.02 (m,2H), 2.16-2.08 (m, 2H); ESI-MS (m/z, %): 273 (MH⁺, 100).

7-Nitro-4-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine

A solution of4-(3-chloropropyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (0.54 g,1.980 mmol) in dry acetonitrile (20 mL) was treated with pyrrolidine(1.637 mL, 19.80 mmol), potassium carbonate (2.74 g, 19.80 mmol), andpotassium iodide (0.657 g, 3.96 mmol) at room temperature. The resultingmixture was stirred at 60° C. overnight (18 hours). The reaction wasbrought to room temperature and diluted with water (50 mL), and theproduct was extracted into ethyl acetate (2×25 mL). The combined ethylacetate layers were washed with brine (20 mL) and then dried (Na₂SO₄).The solvent was evaporated, and the crude material was purified by flashcolumn chromatography (5:95 (2 M NH₃ in MeOH):CH₂Cl₂) to obtain thetitle product (0.57 g, 94%) as a syrup. ¹H NMR (DMSO-d₆) δ 7.84-7.80 (m,2H), 6.87 (d, 1H, J=9.6 Hz), 3.79-3.75 (m, 2H), 3.49 (t, 2H, J=6.9 Hz),3.08-3.05 (m, 2H), 2.44-2.40 (m, 6H), 1.76-1.69 (m, 6H); ESI-MS (m/z,%): 308 (MH⁺, 100).

4-(3-(Pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

A solution of7-nitro-4-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine(0.55 g, 1.789 mmol) in dry methanol (10 mL) was treated with hydrazinehydrate (0.652 mL, 17.89 mmol) followed by Raney-Nickel (0.1 g, 1.789mmol) at room temperature. The resulting mixture was stirred at refluxfor 5 minutes using a pre-heated oil bath. The reaction was then broughtto room temperature, filtered through a pad of Celite, and washed withmethanol (3×10 mL). The combined methanol layers were evaporated, andthe crude material was purified by flash column chromatography (5:95 (2M NH₃ in MeOH):CH₂Cl₂) to obtain the title product (0.45 g, 91%) as asyrup. ¹H NMR (DMSO-d₆) δ 6.51 (d, 1H, J=9.3 Hz), 6.26-6.23 (m, 2H),4.41 (s, 2H), 3.37-3.30 (m, 2H, merged with water peak), 3.11 (t, 2H,J=6.9 Hz), 2.98-2.95 (m, 2H), 2.42-2.37 (m, 6H), 1.67-1.60 (m, 6H).

N-(4-(3-(Pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A solution of4-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.43 g, 1.550 mmol) in dry ethanol (15 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.884 g, 3.10 mmol) at roomtemperature, and the resulting mixture was stirred overnight (18 hours).The reaction was diluted with saturated NaHCO₃ solution (30 mL), and theproduct was extracted into CH₂Cl₂ (3×20 mL). The combined CH₂Cl₂ layerswere washed with brine (20 mL) and then dried (Na₂SO₄). The solvent wasevaporated, and the crude material was purified by column chromatography(5:95 (2 M NH₃ in MeOH):CH₂Cl₂), to obtain the title product 21 (0.51 g,85%) as a solid. ¹H NMR (DMSO-d₆) δ 7.68 (d, 1H, J=4.5 Hz), 7.55 (d, 1H,J=5.1 Hz), 7.06 (dd, 1H, J=3.6, 4.9 Hz), 6.70 (d, 1H, J=8.7 Hz),6.52-6.44 (m, 2H), 6.32 (brs, 2H), 3.51-3.48 (m, 2H), 3.27 (t, 2H, J=7.2Hz), 3.04-3.00 (m, 2H), 2.48-2.40 (m, 6H), 1.80-1.60 (m, 6H); ESI-MS(m/z, %): 387 (MH⁺, 68), 276 (100), 194 (75); HPLC purity: 98.32% byarea.

N-(4-(3-(Pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(3-(pyrrolidin-1-yl)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.48 g, 1.242 mmol) in dry methanol (10 mL) was treated withhydrochloric acid (1 M solution in ether; 3.72 mL, 3.72 mmol) at roomtemperature, and the resulting mixture was stirred for 15 minutes. Thesolvent was then evaporated, and the product was dried under vacuum toobtain the dihydrochloride salt (0.55 g, 96%) as a solid. ¹H NMR(DMSO-d₆) δ 11.34 (brs, 1H), 11.28 (s, 1H), 9.68 (s, 1H), 8.69 (s, 1H),8.16-8.12 (m, 2H), 7.35 (t, 1H, J=4.2 Hz), 3.66-3.60 (m, 2H), 3.50-3.40(m, 4H), 3.16-3.05 (m, 4H), 3.02-2.92 (m, 2H), 2.06-1.84 (m, 2H); ESI-MS(m/z, %): 387 (MH⁺, free base, 92), 276 (87), 194 (100); ESI-HRMScalculated for C₂₀H₂₇N₄S₂ (MH⁺, free base), calculated: 387.1671,observed: 387.1659; HPLC purity: 98.58% by area.

Example 22 Synthesis ofN-(4-(3-(Dimethylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(22)

4-(3-Chloropropyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to reported procedure in Example 21.

N,N-Dimethyl-3-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)propan-1-amine

A solution of4-(3-chloropropyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (0.7 g,2.57 mmol), dimethylamine (2M solution in THF) (2.57 mL, 5.13 mmol),potassium iodide (0.426 g, 2.57 mmol), and potassium carbonate (1.773 g,12.83 mmol) in dry acetonitrile (20 mL) was stirred at 80° C. for 6hours in a sealed tube. The reaction was then brought to roomtemperature and diluted with water (60 mL), and the product wasextracted into ethyl acetate (3×20 mL). The combined ethyl acetatelayers were washed with brine (20 mL) and dried (Na₂SO₄). The solventwas evaporated, and the crude material was purified by columnchromatography (1:9 (2 M NH₃ in MeOH):CH₂Cl₂) on silica gel to obtainthe title product (0.3 g, 42%) as a thick syrup. ¹H NMR (DMSO-d₆) δ7.85-7.80 (m, 2H), 6.85 (d, 1H, J=8.7 Hz), 3.77 (t, 2H, J=5.1 Hz), 3.47(t, 2H, J=7.2 Hz), 3.06 (t, 2H, J=5.1 Hz), 2.24 (t, 2H, J=6.6 Hz), 2.14(s, 6H), 1.75-1.65 (m, 2H); ESI-MS (m/z, %): 282 (MH⁺, 100).

4-(3-(Dimethylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine

A solution ofN,N-dimethyl-3-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)propan-1-amine(0.27 g, 0.960 mmol) in dry methanol (10 mL) was treated with hydrazinehydrate (0.349 mL, 9.60 mmol), followed by Raney-Nickel (0.1 g, 0.096mmol) at room temperature. The resulting mixture was refluxed for 5minutes in a pre-heated oil bath. The reaction was then brought to roomtemperature, filtered through a pad of Celite, and washed with methanol(3×10 mL). The combined methanol layers were evaporated, and the crudematerial was purified by flash column chromatography (5:95 (2 M NH₃ inMeOH):CH₂Cl₂) on silica gel to obtain the title product (0.24 g, 99%) asa syrup. ¹H NMR (DMSO-d₆) δ 6.50 (dd, 1H, J=2.4, 7.0 Hz), 6.26-6.23 (m,2H), 4.41 (s, 2H), 3.32-3.30 (m, 2H), 3.09 (t, 2H, J=7.2 Hz), 2.98-2.95(m, 2H), 2.21 (t, 2H, J=6.9 Hz), 2.11 (s, 6H), 1.63-1.54 (m, 2H); ESI-MS(m/z, %): 252 (MH⁺, 100).

N-(4-(3-(Dimethylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A solution of4-(3-(dimethylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-amine(0.22 g, 0.875 mmol) in dry ethanol (10 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.499 g, 1.750 mmol) at roomtemperature, and the resulting mixture was stirred over night (18 hours)at room temperature. The reaction was diluted with saturated NaHCO₃solution (25 mL), and the product was extracted into CH₂Cl₂ (3×20 mL).The combined CH₂Cl₂ layers were washed with brine (15 mL) and dried(Na₂SO₄). The solvent was evaporated, and the crude material waspurified by column chromatography (5:95 (2M NH₃ in MeOH):CH₂Cl₂) onsilica gel to obtain the title product 22 (0.27 g, 86%) as a solid. ¹HNMR (DMSO-d₆) δ 7.68 (d, 1H, J=3.0 Hz), 7.56 (d, 1H, J=5.1 Hz), 7.06(dd, 1H, J=3.9, 4.8 Hz), 6.69 (d, 1H, J=8.7 Hz), 6.52-6.45 (m, 2H), 6.33(brs, 2H), 3.51-3.48 (m, 2H), 3.25 (t, 2H, J=7.2 Hz), 3.04-3.01 (m, 2H),2.25 (t, 2H, J=6.9 Hz), 2.14 (s, 6H), 1.70-1.61 (m, 2H); ESI-MS (m/z,%): 361 (MH⁺, 93), 276 (100); HPLC purity: 98.23% by area.

N-(4-(3-(Dimethylamino)propyl)-3,4-dihydro-2,1-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(3-(dimethylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.16 g, 0.444 mmol) in dry methanol (5 mL) was treated with hydrogenchloride (1M solution in ether; 1.331 mL, 1.331 mmol) at roomtemperature. The mixture was stirred for 15 minutes, the solvent wasevaporated, and the residue was dried under high vacuum to obtain thetitle product (0.19 g, 99%) as a solid. ¹H NMR (DMSO-d₆) δ 11.22 (s,1H), 10.91 (brs, 1H), 9.67 (s, 1H), 8.69 (s, 1H), 8.15-8.11 (m, 2H),7.36 (t, 1H, J=4.5 Hz), 7.04-6.88 (m, 3H), 3.64-3.61 (m, 2H), 3.41 (t,2H, J=6.9 Hz), 3.14-3.02 (m, 4H), 2.72 (d, 6H, J=4.8 Hz), 2.06-1.98 (m,2H); ESI-MS (m/z, %): 361 (MH⁺, free base, 91), 276 (100); ESI-HRMScalculated for C₁₈H₂₅N₄S₂ (MH⁺, free base), calculated: 361.1515,observed: 361.1515; HPLC purity: 98.15% by area.

Example 23 Synthesis ofN-(4-(3-(methylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(23)

4-(3-Chloropropyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to reported procedure in Example 21.

N-Methyl-3-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)propan-1-amine

A solution of4-(3-chloropropyl)-7-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine (1.1 g,4.03 mmol), methylamine hydrochloride (0.545 g, 8.07 mmol), potassiumiodide (0.669 g, 4.03 mmol), and potassium carbonate (2.79 g, 20.16mmol) in dry acetonitrile (25 mL) was stirred at 80° C. in a sealed tubefor 6 hours. The reaction was brought to room temperature and dilutedwith water (100 mL), and the product was extracted into ethyl acetate(3×25 mL). The combined ethyl acetate layers were washed with brine (20mL) and dried (Na₂SO₄). The solvent was evaporated, and the crudematerial was purified by flash column chromatography (1:9 (2M NH₃ inMeOH):CH₂Cl₂) on silica gel to obtain the title compound (0.8 g, 74%) asa thick syrup. ¹H NMR (DMSO-d₆) δ 7.84-7.81 (m, 2H), 6.86 (d, 1H, J=9.9Hz), 3.78-3.75 (m, 2H), 3.50 (t, 2H, J=7.5 Hz), 3.08-3.04 (m, 2H), 2.48(t, 2H, J=6.9 Hz), 2.27 (s, 3H), 1.74-1.64 (m, 2H); ESI-MS (m/z, %): 268(MH⁺, 100).

tert-Butylmethyl(3-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)propyl)carbamate

A solution ofN-methyl-3-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)propan-1-amine(0.77 g, 2.88 mmol) in dry 1,4-dioxane (20 mL) was treated withtriethylamine (1.214 mL, 8.64 mmol) followed by di-tert-butyldicarbonate (0.736 mL, 3.17 mmol) at room temperature. The reactionstirred for 4 hours. The solvent was then evaporated, and the crudematerial was purified by flash column chromatography (1:1 EtOAc:hexanes)on silica gel to obtain the title product (1.02 g, 96%) as a yellowsolid. ¹H NMR (CDCl₃) δ 7.97 (d, 1H, J=2.4 Hz), 7.88 (dd, 1H, J=2.7, 9.3Hz), 6.56 (d, 1H, J=9.3 Hz), 3.79-3.76 (m, 2H), 3.41 (t, 2H, J=7.8 Hz),3.31 (t, 2H, J=6.9 Hz), 3.04-3.00 (m, 2H), 2.88 (s, 3H), 1.92-1.82 (m,2H), 1.46 (s, 9H); ESI-MS (m/z, %): 390 (M+Na, 56), 368 (MH⁺, 10), 312(60), 268 (100).

tert-Butyl3-(7-amino-2H-benzo[b][1,4]thiazin-4(311)-yl)propylmethyl)carbamate

A suspension of tert-butylmethyl(3-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)propyl)carbamate (0.6g, 1.633 mmol) in dry methanol (10 mL) was treated with Raney-Nickel(0.1 g, 1.633 mmol) followed by hydrazine hydrate (0.595 mL, 16.33 mmol)at room temperature. The resulting mixture was refluxed for 10 minutesin a pre-heated oil bath. The reaction was then brought to roomtemperature, filtered through a pad of Celite, and washed with methanol(3×10 mL). The combined methanol layers were evaporated, and the crudematerial was purified by flash column chromatography (5:95 (2M NH₃ inMeOH):CH₂Cl₂) on silica gel to obtain title product (0.55 g,quantitative) as a brown syrup. ¹H NMR (DMSO-d₆) δ 6.46 (d, 1H, J=9.6Hz), 6.25-6.21 (m, 2H), 4.44 (s, 2H), 3.32-3.28 (m, 2H), 3.19 (t, 2H,J=6.9 Hz), 3.04 (t, 2H, J=7.2 Hz), 3.00-2.97 (m, 2H), 2.77 (s, 3H),1.74-1.62 (m, 2H), 1.38 (s, 9H); ESI-MS (m/z, %): 338 (MH⁺, 100), 337(96), 282 (54).

tert-Butylmethyl(3-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)propyl)carbamate

A solution of tert-butyl3-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)propyl(methyl)carbamate(0.52 g, 1.541 mmol) in dry ethanol (20 mL) was treated with methylthiophene-2-carbimidothioate hydroiodide (0.879 g, 3.08 mmol) at roomtemperature and stirred over night (18 hours). The reaction was basifiedwith saturated NaHCO₃ solution (50 mL), and the product was extractedinto CH₂Cl₂ (2×25 mL). The combined CH₂Cl₂ layers were washed with brine(20 mL) and dried (Na₂SO₄). The solvent was evaporated, and the crudematerial was purified by column chromatography (2:98 (2 M NH₃ inMeOH):CH₂Cl₂) on silica gel to obtain the title product (0.60 g, 87%) asa solid. ESI-MS (m/z, %): 447 (MH⁺, 100).

N-(4-(3-(Methylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A suspension of tert-butylmethyl(3-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)propyl)carbamate(0.55 g, 1.231 mmol) in 3M hydrochloric acid (25 mL) was refluxed for 1hour. The reaction was brought to room temperature, filtered, and washedwith water (2×10 mL). The combined aqueous layers were evaporated. Thecrude material was basified with 3 M NaOH solution (50 mL), and theproduct was extracted into CH₂Cl₂ (3×20 mL). The combined CH₂Cl₂ layerswere washed with brine (15 mL) and dried (Na₂SO₄). The organic solventwas evaporated, and the crude material was purified by columnchromatography (5:95 to 1:9 (2M NH₃ in MeOH):CH₂Cl₂) on silica gel toobtain compound 23 (0.4 g, 94%) as a solid. ¹H NMR (DMSO-d₆) δ 7.68 (dd,1H, J=1.2, 3.7 Hz), 7.56 (dd, 1H, J=0.9, 5.1 Hz), 7.06 (dd, 1H, J=3.6,4.9 Hz), 6.70 (d, 1H, J=8.7 Hz), 6.51-6.44 (m, 2H), 6.31 (s, 2H),3.50-3.47 (m, 2H), 3.32-3.25 (m, 4H), 3.04-3.01 (m, 2H), 2.27 (s, 3H),1.70-1.61 (m, 2H); ESI-MS (m/z, %): 347 (MH⁺, 80), 276 (100); HPLCpurity: 96.05% by area.

N-(4-(3-(Methylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(3-(methylamino)propyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.37 g, 1.068 mmol) in dry methanol (10 mL) was treated with hydrogenchloride (1 M in ether) (3.20 mL, 3.20 mmol) and stirred for 15 minutes.The solvent was evaporated, and the crude material was dried under highvacuum to obtain the dihydrochloride (0.44 g, 98%) as a solid. ¹H NMR(DMSO-d₆) δ 11.22 (s, 1H), 9.66 (s, 1H), 9.19 (brs, 2H), 8.69 (s, 1H),8.15-8.12 (m, 2H), 7.35 (t, 1H, J=4.2 Hz), 7.04-6.89 (m, 3H), 3.64-3.61(m, 2H), 3.44 (t, 2H, J=6.9 Hz), 3.10-3.07 (m, 2H), 2.96-2.86 (m, 2H),2.53 (t, 3H, J=5.7 Hz), 1.98-1.86 (m, 2H); ESI-MS (m/z, %): 347 (MH⁺,free base, 65), 276 (100); ESI-HRMS calculated for C₁₇H₂₃N₄S₂ (MH⁺, freebase), calculated: 347.1358, observed: 347.1345; HPLC purity: 96.11% byarea.

Example 24 Synthesis ofN-(4-(2-(ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(24)

2-(2-Amino-4-nitrophenylthio)ethanol

To a stirred solution of 2-chloro-5-nitroaniline (1.0 g, 5.79 mmol) inDMF (10 mL) was added potassium carbonate (1.60 g, 11.59 mmol) followedby 2-mercaptoethanol (0.813 mL, 11.59 mmol). The resulting mixture wasthen heated at 60° C. for 2 hours and then at room temperatureovernight. The mixture was then diluted with ethyl acetate and washedwith water (3×), 1N NaOH, and brine. The organic phase was dried,filtered, and concentrated, giving a red/orange solid (1.19 g, 96%). ¹HNMR (DMSO-d₆) δ 7.52 (s, 1H), 7.42-7.33 (m, 2H), 5.79 (s, 2H), 4.99 (t,J=5.4 Hz, 1H), 3.55 (q, J=6.2 Hz, 2H), 3.02 (t, J=6.6 Hz, 2H); ESI-MS(m/z, %): 215 (MH⁺, 23), 169 (100), 111 (63).

2-(2-Iodoethylthio)-5-nitroaniline

To a stirred solution of triphenylphosphine (1.81 g, 6.93 mmol) andimidazole (1.887 mL, 13.86 mmol) in THF (15 mL) at 0° C. was addediodine (1.759 g, 6.93 mmol). After stirring for 5 minutes,2-(2-amino-4-nitrophenylthio)ethanol (990 mg, 4.62 mmol) was added as asolution in THF (5 mL). The resulting mixture was stirred at 0° C. for 1hour, then diluted with ethyl acetate and washed with water (3×) andbrine. The organic phase was dried, filtered, and concentrated thenchromatographed in 4:1 hexanes:ethyl acetate giving the desired compound(1.18 g, 79%) as an orange solid. ¹H NMR (DMSO-d₆) δ 7.55 (d, J=2.4 Hz,1H), 7.45 (d, J=8.4 Hz, 1H), 7.34 (dd, J=8.4, 2.4 Hz, 1H), 5.89 (brs,2H), 3.38-3.26 (m, 4H); EI-MS (m/z, %): 324 (M⁺, 46), 196 (81), 154(100), 126 (95).

6-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

To a stirred solution of 2-(2-iodoethylthio)-5-nitroaniline (1.18 g,3.64 mmol) in DMF (10 mL) was added potassium carbonate (1.006 g, 7.28mmol). The resulting mixture was then stirred at 90° C. for 1 hour. Themixture was then cooled to room temperature and diluted with water (40mL). The resulting red precipitate was collected by vacuum filtration,giving the title compound (625 mg, 87%). ¹H NMR (DMSO-d₆) δ 7.38 (s,1H), 7.28 (d, J=8.4 Hz, 1H), 7.12 (d, J=8.7 Hz, 6.78 (brs, 1H),3.56-3.49 (m, 2H), 3.08-3.03 (m, 2H); ELMS (m/z, %): 196 (M⁺, 100), 181(45), 122 (73).

2-Chloro-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

To a stirred solution of 6-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine(620 mg, 3.16 mmol) in THF (10 mL) was added 2-chloroacetyl chloride(0.277 mL, 3.48 mmol). The resulting mixture was then stirred at 60° C.for 10 minutes. The mixture was then diluted with ethyl acetate andwashed with water (3×), 1:1 water:saturated sodium carbonate, and brine.The organic phase was dried, filtered, and concentrated, giving thedesired product (860 mg, 100%). ¹H NMR (DMSO-d₆) δ 8.36 (d, J=2.1 Hz,1H), 7.96 (dd, J=8.7, 2.1 Hz, 1H), 7.52 (d, J=8.7 Hz, 1H), 4.63 (s, 2H),3.99-3.92 (m, 2H), 3.39-3.33 (m, 2H); ESI-MS (m/z, %): 295 (M+Na, 68),273 (MH⁺, 100), 197 (43).

2-(Ethylamino)-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

To a stirred solution of2-chloro-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone (855 mg,3.14 mmol) in dioxane (15 mL) and triethylamine (0.881 mL, 6.27 mmol)was added ethanamine hydrochloride (1.278 g, 15.68 mmol) as a solutionin water (7.50 mL). The resulting mixture was stirred vigorously at roomtemperature over the weekend. The mixture was then diluted with waterand extracted with dichloromethane (2×). The combined organics weredried, filtered, concentrated, and then chromatographed in 1:9 (2M NH₃in methanol):ethyl acetate, giving the desired product (775 mg, 88%). ¹HNMR (DMSO-d₆) δ 8.37 (d, J=2.4 Hz, 1H), 7.93 (dd, J=8.7, 2.4 Hz, 1H),7.50 (d, J=8.7 Hz, 1H), 3.97-3.90 (m, 2H), 3.52 (s, 2H), 3.37-3.33 (m,2H), 2.58-2.50 (m, 2H), 2.07 (brs, 1H), 1.00 (t, J=7.0 Hz, 3H); ESI-MS(m/z, %): 282 (MH⁺, 100).

tert-Butylethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate

To a stirred solution of2-(ethylamino)-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone (770mg, 2.74 mmol) in dioxane (10 mL) and triethylamine (0.769 mL, 5.47mmol) was added di-tert-butyl dicarbonate (627 mg, 2.87 mmol). Theresulting mixture was stirred at room temperature for 30 minutes. Themixture was then diluted with ethyl acetate and washed sequentially withwater and a 1:1 mixture of water and brine. The organic phase was dried,filtered, and concentrated, giving a pale yellow solid (1.02 g, 98%). ¹HNMR (DMSO-d₆) δ 8.33 (s, 1H), 7.94 (d, J=8.4 Hz, 1H), 7.51 (d, J=8.7 Hz,1H), 4.18 (s, 2H), 3.96-3.89 (m, 2H), 3.32-3.19 (m, 4H), 1.38, 1.30 (2s,9H), 1.09-1.00 (m, 3H); ESI-MS (m/z, %): 404 (M+Na, 51), 382 (MH⁺, 36),282 (100).

tert-Butylethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution of tert-butylethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate(1.01 g, 2.65 mmol) in tetrahydrofuran (5 mL) was addedborane-tetrahydrofuran complex (1 M in tetrahydrofuran; 7.94 mL, 7.94mmol). The resulting mixture was stirred at room temperature for 2hours. The mixture was then quenched via the slow dropwise addition (toavoid excessive bubbling) of MeOH (5 mL). The quenched reaction was thendiluted with ethyl acetate and washed with water (2×) and saturatedsodium carbonate (2×). The organic phase was dried, filtered, andconcentrated, giving a red-orange oil (925 mg, 95%). ¹H NMR (DMSO-d₆) δ7.59-7.41 (m, 1H), 7.39-7.32 (m, 1H), 7.24-7.13 (m, 1H), 3.69-3.62 (m,2H), 3.58-3.48 (m, 2H), 3.45-3.35 (m, 2H), 3.28-3.16 (m, 2H), 3.15-3.08(m, 2H), 1.32, 1.24 (2s, 9H), 1.04 (t, J=6.9 Hz, 3H); ESI-MS (m/z, %):390 (M+Na, 70), 368 (MH⁺, 12), 312 (47), 268 (100).

tert-Butyl2-(6-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(ethyl)carbamate

To a stirred solution of tert-butylethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate (925mg, 2.52 mmol) in ethanol (10 mL) was added Raney-Nickel (˜148 mg, 2.52mmol) followed by hydrazine hydrate (1.225 mL, 25.2 mmol). The resultingmixture was stirred vigorously at 50° C. for 10 minutes. The mixture wasthen cooled to room temperature, diluted with ethyl acetate, and thenwashed sequentially with a 1:1 mixture of water and saturated sodiumcarbonate (3×) and saturated sodium carbonate. The organic phase wasdried, filtered, and concentrated giving a red oil (810 mg, 95%); ESI-MS(m/z, %): 338 (MH⁺, 100), 282 (53), 238 (36).

tert-Butylethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution of tert-butyl2-(6-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl(ethyl)carbamate (800mg, 2.371 mmol) in ethanol (8 mL) was added methylthiophene-2-carbimidothioate hydroiodide (1.014 g, 3.56 mmol). Theresulting mixture was stirred at room temperature for 3 hours. Themixture was then diluted with ethyl acetate and washed with saturatedsodium carbonate (3×). The organic phase was dried, filtered,concentrated, and then chromatographed in 1:1 hexanes:ethyl acetategiving the desired product (415 mg, 39.2%). ¹H NMR (DMSO-d₆) δ 7.70 (d,J=3.0 Hz, 1H), 7.58 (d, J=2.4 Hz, 1H), 7.49-7.44 (m, 1H), 7.07 (t, J=4.4Hz, 1H), 6.84 (d, J=7.8 Hz, 1H), 6.35-6.26 (m, 2H), 6.08 (d, J=7.5 Hz,1H), 3.64-3.49 (m, 2H), 3.45-3.25 (m, 4H), 3.24-3.12 (m, 2H), 3.01-2.85(m, 2H), 1.39-1.29 (m, 9H), 1.02 (t, J=6.9 Hz, 3H); ESI-MS (m/z, %): 447(MH⁺, 100).

N-(4-(2-(Ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide

To a stirred suspension of tert-butylethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(410 mg, 0.918 mmol) in methanol (6 mL) was added a 3N HCl solution(3.06 mL, 9.18 mmol). The resulting mixture was stirred at 90° C. for 30minutes. The mixture was then passed through a 0.45 μM syringe filter,and the filtrate was concentrated in vacuo to dry foam. The residue wasthen partitioned between 1:1 water:saturated sodium carbonate anddichloromethane. The organic layer was separated, and the aqueous phasewas extracted again with dichloromethane. The organic phase was dried,filtered, concentrated, and then chromatographed in 1:4:5 (2M NH₃ inmethanol):ethyl acetate:dichloromethane giving the desired product 24(135 mg, 42.4%). ¹H NMR (DMSO-d₆) δ 7.71 (d, J=3.6 Hz, 1H), 7.58 (d, 5.1Hz, 1H), 7.08 (t, J=4.4 Hz, 1H), 6.82 (d, J=7.8 Hz, 1H), 6.33 (brs, 2H),6.21 (s, 1H), 6.07 (dd, J=8.1, 1.5 Hz, 1H), 3.63-3.57 (m, 2H), 3.33-3.28(m, 2H), 3.01-2.96 (m, 2H), 2.71-2.65 (m, 2H), 2.53 (q, J=7.0 Hz, 2H),1.66 (brs, 1H), 1.17 (t, J=7.0 Hz, 3H); ESI-MS (m/z, %): 347 (MH⁺, 83),276 (100); HRMS calculated for C₁₇H₂₃N₄S₂ (MH⁺), calculated: 347.1358,observed: 347.1346; HPLC purity: 97% by area.

N-(4-(2-(Ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a solution ofN-(4-(2-(ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(131 mg, 0.378 mmol) in methanol (2 mL) was added hydrogen chloride (1Min diethyl ether; 1.134 mL, 1.134 mmol). The resulting mixture wasconcentrated in vacuo, giving an orange solid, (159 mg, 100%). ¹H NMR(DMSO-d₆) δ 11.43 (s, 1H), 9.72 (s, 1H), 9.22 (brs, 2H), 8.73 (s, 1H),8.18-8.12 (m, 2H), 7.38-7.34 (m, 1H), 7.14-7.05 (m, 2H), 6.64-6.60 (m,1H), 3.70-3.61 (m, 4H), 3.16-3.02 (m, 4H), 2.99-2.88 (m, 2H), 1.21 (t,J=7.2 Hz, 3H); HRMS (C₁₇H₂₃N₄S₂, MH⁺, free base): calculated: 347.1358,observed: 347.1346. HPLC purity: 97% by area.

Example 25 Synthesis ofN-(4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(25)

2-Chloro-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

Prepared according to the procedure reported in Example 24.

1-(6-Nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-(pyrrolidin-1-yl)ethanone

To a stirred solution of2-chloro-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone (685 mg,2.51 mmol) in dioxane (10 mL), cooled to 0° C. was added HCl, 1 M indiethyl ether (2.51 mL, 2.51 mmol) followed by pyrrolidine (0.21 mL,2.51 mmol) dropwise. The resulting mixture was stirred at 0° C. for 5minutes and then warmed to room temperature, stirring overnight. At thistime, additional pyrrolidine (0.21 mL, 2.51 mmol) was added, followed bywater (1 mL) to aid dissolution. After 1 hour of stirring at roomtemperature, the mixture was then diluted with ethyl acetate and washedwith saturated sodium carbonate, water (3×), and brine. The organicphase was dried, filtered, and concentrated, giving a yellow/orange oil(756 mg, 98%). ¹H NMR (DMSO-d₆) δ 8.52 (brs, 1H), 7.93 (dd, J=9.0, 1.6Hz, 1H), 7.49 (d, J=9.0 Hz, 1H), 4.01-3.91 (m, 4H), 3.56 (s, 2H),3.45-3.35 (m, 2H), 2.55-2.50 (m, 2H), 2.22-2.14 (m, 4H); ESI-MS (m/z,%): 308 (MH⁺, 100).

6-Nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine

To a stirred solution of1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-(pyrrolidin-1-yl)ethanone(750 mg, 2.440 mmol) in tetrahydrofuran (5 mL) was addedborane-tetrahydrofuran complex (1M in tetrahydrofuran; 7.32 mL, 7.32mmol). The resulting mixture was then stirred at 55° C. overnight. Thereaction mixture was then cooled to room temperature and quenchedcarefully via the slow, dropwise addition of methanol (5 mL). Thequenched mixture was then stirred at 55° C. overnight. At this time, thereaction was vented to the atmosphere. The mixture was then concentratedand chromatographed on silica gel, eluting with 1:4:5 (2M NH₃ inmethanol):ethyl acetate:dichloromethane, giving the desired product (654mg, 91%) as a red oil. ¹H NMR (DMSO-d₆) δ 7.46 (d, J=2.4 Hz, 1H), 7.36(dd, J=8.4, 2.1 Hz, 1H), 7.19 (d, J=8.4 Hz, 1H), 3.69-3.65 (m, 2H), 3.50(t, J=6.9 Hz, 2H), 3.14-3.10 (m, 2H), 2.63 (t, J=6.9 Hz, 2H), 2.56-2.51(m, 2H), 1.71-1.66 (m, 4H); ESI-MS (m/z, %): 294 (MH⁺, 100).

N-(4-(2-(Pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide

To a stirred solution of6-nitro-4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazine(649 mg, 2.212 mmol) in ethanol (5 mL) was added palladium, 10 wt. % onactivated carbon (235 mg, 0.221 mmol) as a suspension in ethanol (5 mL).The resulting suspension was stirred under an atmosphere of hydrogen(balloon pressure) for 3 hours (yellow color disappears). The balloonwas removed, and methyl thiophene-2-carbimidothioate hydroiodide (1.262g, 4.42 mmol) was added to the mixture. The resulting mixture wasstirred under argon overnight at room temperature. The mixture was thendiluted with dichloromethane and filtered through Celite. The filtratewas then diluted with saturated sodium carbonate and extracted withdichloromethane (2×). The combined organics were dried, filtered,concentrated, and then chromatographed in 1:19 (2M NH₃ inmethanol):ethyl acetate, giving the desired product 25 (438 mg, 53.1%).¹H NMR (DMSO-d₆) δ 7.71 (d, J=3.3 Hz, 1H), 7.58 (d, J=5.1 Hz, 1H),7.10-7.06 (m, 1H), 6.83 (d, J=8.1 Hz, 1H), 6.38 (brs, 2H), 6.19 (s, 1H),6.08 (d, J=1H), 3.63-3.58 (m, 2H), 3.40-3.33 (m, 2H), 3.01-2.97 (m, 2H),2.61 (t, J=6.8 Hz, 2H), 2.49-2.42 (m, 4H), 1.70-1.61 (m, 4H); ESI-MS(m/z, %): 373 (MH⁺, 62), 276 (100); HRMS calculated for C₁₉H₂₅N₄S₂(MH⁺), calculated: 373.1515, observed: 373.1512; HPLC purity: 97% byarea.

N-(4-(2-(Pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a solution ofN-(4-(2-(pyrrolidin-1-yl)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(424 mg, 1.138 mmol) in methanol (4 mL) was added hydrogen chloride (1 Min diethyl ether; 3.41 mL, 3.41 mmol). The resulting mixture wasconcentrated in vacuo, giving an orange solid (507 mg, 100%). ¹H NMR(DMSO-d₆) δ 11.60-11.40 (m, 2H), 9.75 (s, 1H), 8.78 (s, 1H), 8.22-8.10(m, 2H), 7.36 (t, J=8.4 Hz, 1H), 7.15-7.05 (m, 2H), 6.64 (d, J=8.1 Hz,1H), 3.80-3.71 (m, 2H), 3.70-3.61 (m, 2H), 3.58-3.45 (m, 2H), 3.43-3.30(m, 2H), 3.16-3.07 (m, 2H), 3.06-2.95 (m, 2H), 2.05-1.92 (m, 2H),1.90-1.79 (m, 2H); HRMS (C₁₉H₂₅N₄S₂, MH⁺, free base): calculated:373.1515, observed: 373.1512. HPLC purity: 97% by area.

Example 26 Synthesis ofN-(4-(2-(2-hydroxyethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(26)

6-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the reported procedure in Example 24.

2-(2-Hydroxyethylamino)-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

To a stirred solution of 6-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine(500 mg, 2.55 mmol) in tetrahydrofuran (10 mL) was added 2-chloroacetylchloride (302 mg, 2.68 mmol). The resulting mixture was stirred at 60°C. for 5 minutes. The reaction first turns cloudy then clarifies. Atthis time, the reaction was cooled to 0° C., and hydrogen chloride (4 Min dioxane; 5.10 mL, 20.38 mmol) and 2-aminoethanol (1.868 g, 30.6 mmol)were added to the reaction simultaneously in a dropwise fashion. Theresulting mixture was warmed to room temperature, and water (5 mL) wasadded to aid dissolution. The mixture was then stirred at 60° C. for 3hours. The mixture was then diluted with water and saturated sodiumcarbonate and then extracted with dichloromethane (5×). The combinedorganic layers were dried, filtered, concentrated, and thenchromatographed in 1:4:5 (2M NH₃ in methanol):ethylacetate:dichloromethane giving the desired product (473 mg, 62.4%) as apale yellow solid. ¹H NMR (DMSO-d₆) δ 8.36 (d, J=2.1 Hz, 1H), 7.93 (dd,J=8.7, 2.1 Hz, 1H), 7.50 (d, J=9.0 Hz, 1H), 4.50 (t, J=5.1 Hz, 1H),3.96-3.90 (m, 2H), 3.56 (s, 2H), 3.48-3.40 (m, 2H), 3.32-3.26 (m, 2H),2.58 (t, J=5.0 Hz, 2H), 2.18-2.08 (brs, 1H); ESI-MS (m/z, %): 298 (MH⁺,100).

tert-Butyl2-hydroxyethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate

To a stirred solution of2-(2-hydroxyethylamino)-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone(470 mg, 1.581 mmol) in dioxane (10 mL) and triethylamine (0.444 mL,3.16 mmol) was added di-tert-butyl dicarbonate (362 mg, 1.660 mmol). Theresulting mixture was stirred for 30 minutes at room temperature. Themixture was then diluted with ethyl acetate and washed three times witha 1:1 mixture of water and saturated sodium carbonate. The organic phasewas dried, filtered, and concentrated giving a pale yellow foam (625 mg,99%). ¹H NMR (DMSO-d₆) δ 8.35-8.30 (m, 1H), 7.99-7.91 (m, 1H), 7.56-7.48(m, 1H), 4.67-4.59 (m, 1H), 4.26-4.21 (m, 2H), 3.96-3.88 (m, 2H),3.51-3.43 (m, 2H), 3.31-3.21 (m, 4H), 1.38, 1.31 (2s, 9H); ESI-MS (m/z,%): 420 (M+Na, 47), 398 (MH⁺, 34), 320 (35), 298 (100).

tert-Butyl2-hydroxyethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution of tert-butyl2-hydroxyethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate(620 mg, 1.560 mmol) in tetrahydrofuran (5 mL) was addedborane-tetrahydrofuran complex (1 M in tetrahydrofuran; 4.68 mL, 4.68mmol), dropwise to avoid excessive bubbling. The mixture was thenstirred at room temperature for 2 hours. The mixture was then quenchedvia the slow, dropwise addition of MeOH. The quenched reaction was thendiluted with ethyl acetate and washed with saturated sodium carbonate(3×). The organic phase was dried, filtered, and concentrated giving ared oil (595 mg, 99%). ¹H NMR (DMSO-d₆) δ 7.59-7.44 (m, 1H), 7.38-7.32(m, 1H), 7.22-7.13 (m, 1H), 4.74 (t, J=5.1 Hz, 1H), 3.69-3.62 (m, 2H),3.61-3.39 (m, 6H), 3.29-3.17 (m, 2H), 3.15-3.04 (m, 2H), 1.31, 1.24 (2s,9H); ESI-MS (m/z, %): 406 (M+Na, 55), 328 (40), 284 (100).

tert-Butyl2-hydroxyethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

A stirred suspension of tert-butyl2-hydroxyethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(590 mg, 1.539 mmol) and palladium, 10 wt. % on activated carbon (164mg, 0.154 mmol) in ethanol (10 mL) was stirred at room temperature underan atmosphere of hydrogen (1 atm) for 2 hours (yellow color disappears).To this mixture was then added methyl thiophene-2-carbimidothioatehydroiodide (878 mg, 3.08 mmol), and the resulting mixture was stirredovernight at room temperature. The mixture was then diluted withdichloromethane and then filtered through Celite. The filtrate was thendiluted with water and extracted with dichloromethane (4×). The combinedorganics were dried, filtered, concentrated, and then chromatographed inethyl acetate, giving the desired product (339 mg, 47.6%) as a yellowfoam. ¹H NMR (DMSO-d₆) δ 7.69 (s, 1H), 7.58 (d, J=4.8 Hz, 1H), 7.07 (t,J=4.2 Hz, 1H), 6.41-6.25 (m, 3H), 6.08 (d, J=8.1 Hz, 1H), 4.71-4.65 (m,1H), 3.63-3.54 (m, 2H), 3.49-3.33 (m, 6H), 3.27-3.15 (m, 2H), 3.02-2.94(m, 2H), 1.39-1.29 (m, 9H); ESI-MS (m/z, %): 463 (MH⁺, 100).

N-(4-(2-(2-Hydroxyethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide

To a stirred solution of tert-butyl2-hydroxyethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(334 mg, 0.722 mmol) in methanol (5 mL) was added HCl, 3M in water(2.407 mL, 7.22 mmol). The resulting solution was stirred at 90° C. for1 hour. The mixture was then cooled to room temperature, diluted withsaturated sodium carbonate and extracted with dichloromethane (6×). Thecombined organics were dried, filtered, concentrated, and thenchromatographed in 1:9 (2M NH₃ in methanol):ethyl acetate, giving thedesired product 26 (175 mg, 66.9%). ¹H NMR (DMSO-d₆) δ 7.71 (d, J=3.3Hz, 1H), 7.58 (d, J=5.1 Hz, 1H), 7.10-7.06 (m, 1H), 6.83 (d, J=8.1 Hz,1H), 6.34 (brs, 2H), 6.22 (s, 1H), 6.07 (d, J=7.5 Hz, 1H), 4.49-4.43 (m,1H), 3.63-3.55 (m, 2H), 3.46-3.38 (m, 2H), 3.37-3.28 (m, 2H), 3.01-2.95(m, 2H), 2.71 (t, J=6.4 Hz, 2H), 2.59 (t, J=5.7 Hz, 2H), 1.91-1.75 (m,1H); ESI-MS (m/z, %): 363 (MH⁺, 100), 276 (99), 268 (62); HRMScalculated for C₁₇H₂₃N₄OS₂ (MH⁺), calculated: 363.1307, observed:363.1290; HPLC purity: 97% by area.

N-(4-(2-(2-Hydroxyethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a solution ofN-(4-(2-(2-hydroxyethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(165 mg, 0.455 mmol) in methanol (2 mL) was added HCl (1M in diethylether; 1.365 mL, 1.365 mmol). The resulting solution was concentrated,giving a yellow solid (197 mg, 99%). ¹H NMR (DMSO-d₆) δ 11.46 (s, 1H),9.75 (s, 1H), 9.21-9.09 (m, 2H), 8.74 (s, 1H), 8.21-8.11 (m, 2H),7.40-7.32 (m, 1H), 7.12 (d, J=8.1 Hz, 1H), 7.03 (s, 1H), 6.62 (d, J=7.8Hz, 1H), 3.73-3.60 (m, 6H), 3.18-3.07 (m, 4H), 3.06-2.98 (m, 2H); HRMS(C₁₇H₂₃N₄OS₂, MH⁺, free base): calculated: 363.1307, observed: 363.1290.HPLC purity: 97% by area.

Example 27 Synthesis ofN-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(27)

6-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the reported procedure in Example 24.

N,N-Dimethyl-2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine

To a stirred suspension of 6-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine(500 mg, 2.55 mmol), 2-chloro-N,N-dimethylethanamine hydrochloride (734mg, 5.10 mmol), and tetrabutylammonium bromide (41.1 mg, 0.127 mmol) indichloromethane (5 mL) was added 50% aqueous NaOH (5 mL). The reactionvessel was then sealed, and the mixture was stirred vigorously overnightat room temperature. The mixture was then diluted with water andextracted with dichloromethane (3×). The combined organics were dried,filtered, concentrated, and then chromatographed in 1:4:5 (2M NH₃ inmethanol):ethyl acetate:dichloromethane, giving the desired product (80mg, 11.74%) and significant amounts of starting material. ¹H NMR(DMSO-d₆) δ 7.44 (d, J=2.4 Hz, 1H), 7.36 (dd, J=8.4, 2.4 Hz, 1H), 7.19(d, J=8.4 Hz, 1H), 3.69-3.64 (m, 2H), 3.48 (t, J=6.9 Hz, 2H), 3.14-3.09(m, 2H), 2.45 (t, J=6.9 Hz, 2H), 2.22 (s, 6H); ESI-MS (m/z, %): 268(MH⁺, 100).

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide

A suspension ofN,N-dimethyl-2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanamine (75mg, 0.281 mmol) and palladium, 10 wt. % on activated carbon (29.9 mg,0.028 mmol) in ethanol (5 mL) was stirred at room temperature under anatmosphere of hydrogen (balloon pressure) for 90 minutes. During thistime, the yellow color of the reaction dissipated. To this mixture wasthen added methyl thiophene-2-carbimidothioate hydroiodide (160 mg,0.561 mmol), and the resulting suspension was stirred overnight at roomtemperature. The mixture was then diluted with dichloromethane andfiltered to remove palladium. The organic filtrate was further dilutedwith water and saturated sodium carbonate (1:1). The organic layer wasseparated, and the aqueous layer was extracted again withdichloromethane. The combined organics were dried, filtered,concentrated, and then chromatographed in 1:19 (2M NH₃ inmethanol):ethyl acetate, giving the desired product 27 (61 mg, 62.8%).¹H NMR (DMSO-d₆) δ 7.71 (d, J=3.3 Hz, 1H), 7.58 (d, J=5.1 Hz, 1H),7.10-7.06 (m, 1H), 6.83 (d, J=8.1 Hz, 1H), 6.37 (brs, 2H), 6.17 (s, 1H),6.08 (d, J=7.8 Hz, 1H), 3.63-3.58 (m, 2H), 3.39-3.32 (m, 2H), 3.01-2.96(m, 2H), 2.42 (t, J=6.9 Hz, 2H), 2.10 (s, 6H); ESI-MS (m/z, %): 347(MH⁺, 100), 276 (74); ESI-HRMS calculated for C₁₇H₂₃N₄S₂ (MH⁺),calculated: 347.1358, observed: 347.1349; HPLC purity: 95% by area.

N-(4-(2-(Dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a solution ofN-(4-(2-(dimethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(57 mg, 0.164 mmol) in methanol (2 mL) was added hydrogen chloride (1 Min diethyl ether; 0.493 mL, 0.493 mmol). The resulting mixture was thenconcentrated in vacuo to give a yellow-orange solid (68 mg, 99%). ¹H NMR(DMSO-d₆) δ 11.49 (s, 1H), 11.09 (brs, 1H), 9.74 (s, 1H), 8.78 (s, 1H),8.19-8.12 (m, 2H), 7.39-7.33 (m, 1H), 7.15-7.05 (m, 2H), 6.66-6.60 (m,1H), 3.79-3.70 (m, 2H), 3.70-3.60 (m, 2H), 3.32-3.21 (m, 2H), 3.16-3.08(m, 2H), 2.81-2.74 (m, 6H); HRMS (C₁₇H₂₃N₄S₂, MH⁺, free base):calculated: 347.1358, observed: 347.1349. HPLC purity: 95% by area.

Example 28 Synthesis ofN-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(28)

6-Nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine

Prepared according to the reported procedure in Example 24.

2-(Methylamino)-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

To a stirred solution of 6-nitro-3,4-dihydro-2H-benzo[b][1,4]thiazine(500 mg, 2.55 mmol) in tetrahydrofuran (4 mL) was added 2-chloroacetylchloride (0.223 mL, 2.80 mmol). The resulting mixture was stirred at 60°C. for 10 minutes, at which time the reaction was cooled to 0° C. To themixture was then added methylamine (2 M in tetrahydrofuran; 12.7 mL,25.4 mmol). The resulting mixture was stirred at room temperature for 30minutes. The mixture was then diluted with water and saturated sodiumcarbonate then extracted with dichloromethane (3×). The combinedorganics were dried, filtered, concentrated, and then chromatographed in1:4:5 (2M NH₃ in methanol:ethyl acetate:dichloromethane, giving thedesired product (511 mg, 75%) as a yellow solid. ¹H NMR (DMSO-d₆) δ 8.36(d, J=2.4 Hz, 1H), 7.93 (dd, J=8.7, 2.4 Hz, 1H), 7.50 (d, J=9.0 Hz, 1H),3.93 (t, J=5.1 Hz, 2H), 3.48 (s, 2H), 3.34-3.28 (m, 2H), 2.28 (s, 3H),2.00 (brs, 1H); ESI-MS (m/z, %): 268 (MH⁺, 100).

tert-Butylmethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate

To a stirred solution of2-(methylamino)-1-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone(506.5 mg, 1.895 mmol) in dioxane (10 mL) and triethylamine (0.533 mL,3.79 mmol) was added di-tert-butyl dicarbonate (434 mg, 1.990 mmol), andthe resulting mixture was then stirred at room temperature for 30minutes. The mixture was then diluted with ethyl acetate and washed withwater (3×) and brine. The organic phase was dried, filtered, andconcentrated giving the desired product (695 mg, 100%) as a pale foam.¹H NMR (DMSO-d₆) δ 8.36-8.28 (m, 1H), 7.98-7.92 (m, 1H), 7.56-7.48 (m,1H), 4.23-4.16 (m, 2H), 3.96-3.88 (m, 2H), 3.33-3.28 (m, 2H), 2.84 (m,3H), 1.39. 1.30 (2s, 9H); ESI-MS (m/z, %): 390 (M+Na, 100), 368 (MH⁺,16), 312 (35), 268 (100).

tert-Butylmethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

To a stirred solution of tert-butylmethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate(691 mg, 1.881 mmol) in tetrahydrofuran (5 mL) was addedborane-tetrahydrofuran complex (1 M in tetrahydrofuran; 5.64 mL, 5.64mmol). The resulting mixture was then stirred at room temperature for 2hours and quenched carefully via the slow, dropwise addition of methanol(5 mL). The quenched mixture was then diluted with ethyl acetate andwashed sequentially with a 1:1 mixture of water:saturated sodiumcarbonate (2×) and then with brine. The organic phase was dried,filtered, and concentrated giving a red oil (659 mg, 99%). ¹H NMR(DMSO-d₆) δ 7.56-7.39 (m, 1H), 7.39-7.31 (m, 1H), 7.24-7.14 (m, 1H),3.68-3.61 (m, 2H), 3.60-3.49 (m, 2H), 3.48-3.36 (m, 2H), 3.13-3.05 (m,2H), 2.83 (s, 3H), 1.30, 1.17 (2s, 9H); ESI-MS (m/z, %): 376 (M+Na, 85),354 (MH⁺, 24), 298 (100).

tert-Butylmethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(311)-yl)ethyl)carbamate

A stirred suspension of tert-butylmethyl(2-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate (650mg, 1.839 mmol) and palladium, 10 wt. % on activated carbon (196 mg,0.184 mmol) in ethanol (10 mL) was stirred under an atmosphere ofhydrogen (balloon pressure) at room temperature for 2 hours. To themixture was then added methyl thiophene-2-carbimidothioate hydroiodide(787 mg, 2.76 mmol), and the resulting suspension was stirred at roomtemperature overnight. The mixture was filtered to remove palladium,diluted with water and saturated sodium carbonate, and then extractedwith dichloromethane (3×). The combined organics were dried, filtered,concentrated, and then chromatographed in 2:3 ethyl acetate:hexanes toafford the desired product (420 mg, 52.8%). ¹H NMR (DMSO-d₆) δ 7.72-7.68(m, 1H), 7.58 (d, J=5.1 Hz, 1H), 7.09-7.05 (m, 1H), 6.84 (d, J=7.8 Hz,1H), 6.38-6.21 (m, 3H), 6.08 (d, J=8.4 Hz, 1H), 3.64-3.56 (m, 2H),3.42-3.33 (m, 4H), 3.03-2.94 (m, 2H), 2.81 (s, 3H), 1.36-1.22 (m, 9H);ESI-MS (m/z, %): 433 (MH⁺, 100).

N-(4-(2-(Methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide

To a stirred solution of tert-butylmethyl(2-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(415 mg, 0.959 mmol) in methanol (4 mL) was added a 3N HCl solution(3.198 mL, 9.59 mmol). The resulting mixture was then stirred at 90° C.for 1 hour. The mixture was then cooled to room temperature, dilutedwith water, basified with 3N sodium hydroxide to pH 12, and extractedwith dichloromethane (3×). The combined organics were dried, filtered,and concentrated then chromatographed in 1:9 (2M NH₃ in methanol):ethylacetate, giving the desired product 28 (165 mg, 51.7%). ¹H NMR (DMSO-d₆)δ 7.71 (d, J=3.0 Hz, 1H), 7.58 (d, J=5.1 Hz, 1H), 7.10-7.06 (m, 1H),6.82 (d, J=8.1 Hz, 1H), 6.34 (brs, 2H), 6.22 (s, 1H), 6.07 (d, J=8.1 Hz,1H), 3.62-3.57 (m, 2H), 3.32 (t, J=6.8 Hz, 2H), 3.01-2.97 (m, 2H), 2.64(t, J=6.8 Hz, 1H), 2.29 (s, 3H). ESI-MS (m/z, %): 333 (MH⁺, 100), 276(71); ESI-HRMS calculated for C₁₆H₂₁N₄S₂ (MH⁺), calculated: 333.1202,observed: 333.1207; HPLC purity: 97% by area.

N-(4-(2-(Methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a solution ofN-(4-(2-(methylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(158 mg, 0.475 mmol) in methanol (3 mL) was added hydrogen chloride (1Min diethyl ether; 1.426 mL, 1.426 mmol). The resulting mixture wasconcentrated in vacuo, giving a yellow solid (192 mg, 100%). ¹H NMR(DMSO-d₆) δ 11.47 (s, 1H), 9.76 (s, 1H), 9.31-9.19 (m, 2H), 8.74 (s,1H), 8.20-8.11 (m, 2H), 7.39-7.33 (m, 1H), 7.11 (d, J=8.1 Hz, 1H), 7.04(s, 1H), 6.62 (d, J=7.8 Hz, 1H), 3.71-3.61 (m, 4H), 3.18-3.04 (m, 4H),2.58-2.52 (m, 3H); HRMS (C₁₆H₂₁N₄S₂, MH⁺, free base): calculated:333.1202, observed: 333.1207. HPLC purity: 97% by area.

Example 29 Synthesis ofN-(4-(piperidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(29)

tert-Butyl 4-(2-chloro-5-nitrophenylamino)piperidine-1-carboxylate

A solution of 2-chloro-5-nitroaniline (1 g, 5.79 mmol) and tert-butyl4-oxopiperidine-1-carboxylate (2.309 g, 11.59 mmol) in dichloroethane(15 mL) and acetic acid (0.995 mL, 17.38 mmol) was stirred at roomtemperature for 30 minutes. At this time, the reaction was treated withsodium triacetoxyborohydride (3.07 g, 14.49 mmol), and the resultingmixture was stirred over the weekend. Additional tert-butyl4-oxopiperidine-1-carboxylate (2.309 g, 11.59 mmol), sodiumtriacetoxyborohydride (3.07 g, 14.49 mmol), and dichloroethane (15 mL)were added. Stirring was continued for 2 days. The mixture was thendiluted with water and 1N sodium hydroxide (basified to pH 10) and thenextracted with dichloromethane (3×). The combined organics were dried,filtered, concentrated, and then chromatographed in 1:19 ethylacetate:hexanes, giving a yellow/orange foam (1.26 g, 61.1%). ¹H NMR(DMSO-d₆) δ 7.54 (d, J=8.7 Hz, 1H), 7.51 (d, J=2.4 Hz, 1H), 7.39 (dd,J=8.7, 2.4 Hz, 1H), 5.76-5.63 (m, 1H), 3.99-3.90 (m, 2H), 3.74-3.62 (m,1H), 3.05-2.80 (m, 2H), 1.89-1.80 (m, 2H), 1.49-1.32 (m, 11H); ESI-MS(m/z, %): 378 (M+Na, 100), 356 (MH⁺, 40), 300 (62).

tert-Butyl4-(2-(2-hydroxyethylthio)-5-nitrophenylamino)piperidine-1-carboxylate

To a stirred solution of tert-butyl4-(2-chloro-5-nitrophenylamino)piperidine-1-carboxylate (1.25 g, 3.51mmol) in DMF (10 mL) were added sequentially potassium carbonate (0.971g, 7.03 mmol) and 2-mercaptoethanol (0.493 mL, 7.03 mmol). The resultingmixture was stirred at 60° C. for 1 hour, after which TLC analysis (2:3ethyl acetate:hexanes) showed that the reaction was complete. Themixture was then diluted with ethyl acetate and washed sequentially withwater and saturated sodium carbonate (3×). The organic phase was dried,filtered, concentrated, and then chromatographed in 2:3 ethylacetate:hexanes, giving the desired product (1.202 g, 86%) as an orangefoam. ¹H NMR (DMSO-d₆) δ 7.51 (d, J=8.4 Hz, 1H), 7.43-7.37 (m, 2H), 5.40(d, J=8.1 Hz, 1H), 5.06 (t, J=5.4 Hz, 1H), 3.95-3.86 (m, 2H), 3.75-3.60(m, 1H), 3.59-3.50 (m, 2H), 3.01-2.85 (m, 4H), 1.94-1.83 (m, 2H),1.49-1.29 (m, 11H); ESI-MS (m/z, %): 420 (M+Na, 22), 342 (100).

tert-Butyl4-(2-(2-iodoethylthio)-5-nitrophenylamino)piperidine-1-carboxylate

To a stirred solution of triphenylphosphine (1.178 g, 4.49 mmol) andimidazole (0.611 g, 8.98 mmol) in tetrahydrofuran (10 mL) at 0° C. wasadded iodine (1.292 g, 5.09 mmol). The resulting dark mixture wasstirred at 0° C. After 5 minutes, tert-butyl4-(2-(2-hydroxyethylthio)-5-nitrophenylamino)piperidine-1-carboxylate(1.19 g, 2.99 mmol) was added as a solution in tetrahydrofuran (5 mL).The mixture was then warmed to room temperature and stirred for 1 hour.The mixture was then diluted with ethyl acetate and washed withsaturated sodium carbonate, saturated sodium thiosulfate, water, andbrine. The organic phase was dried, filtered, concentrated, and thenchromatographed in 1:2 ethyl acetate:hexanes, giving the desired product(1.50 g, 99%) as an orange foam. ¹H NMR (DMSO-d₆) δ 7.59-7.54 (m, 1H),7.45-7.38 (m, 2H), 5.45-5.37 (m, 1H), 3.98-3.85 (m, 2H), 3.73-3.61 (m,1H), 3.35-3.27 (m, 5H), 3.19-3.03 (m, 1H), 3.02-2.82 (m, 2H), 1.92-1.84(m, 2H), 1.45-1.34 (m, 11H); ESI-MS (m/z, %): 530 (M+Na, 6), 452 (25),279 (100).

tert-Butyl4-(6-nitro-2H-benzo[b][1,4]thiazin-4(311)-yl)piperidine-1-carboxylate

To a stirred solution of tert-butyl4-(2-(2-iodoethylthio)-5-nitrophenylamino)piperidine-1-carboxylate (1.49g, 2.94 mmol) in DMF (10 mL) was added potassium carbonate (0.812 g,5.87 mmol). The resulting mixture was stirred at 90° C. for 3 hours. Thereaction mixture was then diluted with ethyl acetate and washedsequentially with water and saturated sodium carbonate (2×). The organicphase was dried, filtered, concentrated, and then chromatographed in 1:4ethyl acetate:hexanes, giving the desired product (473 mg, 42.4%) as anorange foam. ¹H NMR (DMSO-d₆) δ 7.57 (d, J=2.4 Hz, 1H), 7.41 (dd, J=8.7,2.4 Hz, 1H), 7.22 (d, J=8.7 Hz, 1H), 4.09-3.88 (m, 3H), 3.47-3.42 (m,2H), 3.12-3.08 (m, 2H), 3.01-2.85 (m, 2H), 1.72-1.51 (m, 4H), 1.41 (s,9H); ESI-MS (m/z, %): 402 (M+Na, 95), 380 (MH⁺, 28), 324 (100), 280(55).

tert-Butyl4-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate

A suspension of tert-butyl4-(6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate(468 mg, 1.233 mmol) and palladium, 10 wt. % on activated carbon (131mg, 0.123 mmol) in ethanol (10 mL) and tetrahydrofuran (1 mL) wasstirred under an atmosphere of hydrogen (balloon pressure) for 2 hours.During this time, the reaction mixture turned colorless. To the mixturewas then added methyl thiophene-2-carbimidothioate hydroiodide (528 mg,1.850 mmol), and the resulting suspension was stirred at roomtemperature overnight. The mixture was then diluted with ethyl acetateand filtered through a pad of Celite. The filtrate was washed withsaturated sodium carbonate (3×). The organic phase was dried, filtered,concentrated, and then chromatographed in 1:5 ethylacetate:dichloromethane to give the desired product (403 mg, 71.2%). ¹HNMR (DMSO-d₆) δ 7.72 (d, J=3.0 Hz, 1H), 7.59 (d, J=5.1 Hz, 1H),7.10-7.06 (m, 1H), 6.86 (d, J=8.1 Hz, 1H), 6.42-6.28 (m, 3H), 6.13 (d,J=8.1 Hz, 1H), 4.06-3.95 (m, 2H), 3.85-3.76 (m, 1H), 3.40-3.33 (m, 2H),2.99-2.93 (m, 2H), 2.92-2.75 (m, 2H), 1.69-1.48 (m, 4H), 1.39 (s, 9H);ESI-MS (m/z, %): 459 (MH⁺, 100).

N-(4-(Piperidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide

To a stirred solution of tert-butyl4-(6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate(398 mg, 0.868 mmol) in MeOH (6 mL) was added aqueous hydrogen chloride,3M (2.893 mL, 8.68 mmol). The resulting solution was stirred at 90° C.for 45 minutes. The mixture was then cooled to room temperature,basified (pH 12) with saturated sodium carbonate, and then extractedwith dichloromethane (3×). The combined organics were dried, filtered,concentrated, and then chromatographed in 1:6 (2M NH₃ in MeOH):ethylacetate to give the desired product 29 (216 mg, 69.4%) as a pale yellowfoam. ¹H NMR (DMSO-d₆) δ 7.71 (d, J=2.7 Hz, 1H), 7.59 (d, J=4.8 Hz, 1H),7.08 (dd, J=5.1, 3.6 Hz, 1H), 6.86 (d, J=8.1 Hz, 1H), 6.37 (brs, 2H),6.26 (s, 1H), 6.13-6.08 (m, 1H), 3.68-3.52 (m, 1H), 3.43-3.38 (m, 2H),3.01-2.93 (m, 4H), 2.61-2.51 (m, 2H), 1.64-1.52 (m, 4H); ESI-MS (m/z,%): 359 (MH⁺, 100); ESI-HRMS calculated for C₁₈H₂₃N₄S₂ (MH⁺),calculated: 359.1358, observed: 359.1351; HPLC purity: 97% by area.

N-(4-(Piperidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a solution ofN-(4-(piperidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(210 mg, 0.586 mmol) in methanol (4 mL) was added hydrogen chloride (1 Min diethyl ether; 1.757 mL, 1.757 mmol). The resulting solution wasconcentrated in vacuo to give a pale yellow solid (252 mg, 100%). ¹H NMR(DMSO-d₆) δ 11.45 (s, 1H), 9.76 (s, 1H), 9.22 (s, 2H), 8.75 (s, 1H),7.40-7.34 (m, 1H), 7.13 (d, J=8.1 Hz, 1H), 7.02 (s, 1H), 6.64 (d, J=8.1Hz, 1H), 4.02-3.92 (m, 1H), 3.46-3.39 (m, 2H), 3.37-3.26 (m, 2H),3.12-2.91 (m, 4H), 2.15-1.98 (m, 2H), 1.86-1.78 (m, 2H); HRMS(C₁₈H₂₃N₄S₂, MH⁺, free base): calculated: 359.1358, observed: 359.1351.HPLC purity: 97% by area.

Example 30 Synthesis ofN-(4-(pyrrolidin-3-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(30)

2-(2-Chloroethylthio)aniline

To a stirred solution of 2-aminobenzenethiol (0.513 mL, 4.79 mmol) inethanol (5 mL) was added sodium hydroxide (192 mg, 4.79 mmol) as asolution in water (5.00 mL). To the resulting mixture was added1-bromo-2-chloroethane (1.197 mL, 14.38 mmol), and the resulting mixturewas stirred vigorously for 20 minutes The mixture was then diluted withethyl acetate and washed with saturated sodium carbonate (3×). Theorganic phase was dried, filtered, and concentrated, giving a yellowliquid, 2-(2-chloroethylthio)aniline (875 mg, 97%). ¹H NMR (DMSO-d₆) δ7.27 (d, J=7.5 Hz, 1H), 7.10-7.04 (m, 1H), 6.73 (d, J=8.1 Hz, 1H),6.56-6.49 (m, 1H), 5.41 (brs, 2H), 3.62 (t, J=7.4 Hz, 2H), 3.02 (t,J=7.4 Hz, 2H). ESI-MS (m/z, %): 188 (MH⁺, 100).

3,4-Dihydro-2H-benzo[b][1,4]thiazine

To a stirred solution of 2-(2-chloroethylthio)aniline (850 mg, 4.53mmol) in DMF (10 mL) was added potassium carbonate (1878 mg, 13.59 mmol)followed by sodium iodide (67.9 mg, 0.453 mmol). The resulting mixturewas heated to 90° C. and stirred overnight. The reaction mixture wasthen cooled to room temperature, diluted with ethyl acetate, and washedwith water (3×) and brine. The organic phase was dried, filtered,concentrated, and then chromatographed in 9:1 hexanes:ethyl acetate,giving an orange oil (675 mg, 99%). ¹H NMR (DMSO-d₆) δ 6.85-6.75 (m,2H), 6.51-6.39 (m, 2H), 6.00 (brs, 1H), 3.49-3.44 (m, 2H), 2.98-2.93 (m,2H); ESI-MS (m/z, %): 152 (MH⁺, 100), 124 (44).

tert-Butyl 3-(2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate

A mixture of 3,4-dihydro-2H-benzo[b][1,4]thiazine (1.00 g, 6.61 mmol),N-boc-3-pyrrolidinone (1.34 g, 7.27 mmol), and acetic acid (0.94 mL,16.52 mmol) in 1,2-dichloroethane (20 mL) was cooled to 0° C. and thentreated with solid sodium triacetoxyborohydride (2.10 g, 9.92 mmol). Thereaction was brought to room temperature and was stirred for 11 hours.Additional equivalents of each of sodium triacetoxyborohydride (1.40 g,6.61 mmol) and N-boc-3-pyrrolidinone (1.22 g, 6.61 mmol) were added tothe reaction mixture. The reaction was stirred for 2 more days and wasthen quenched with 3 N NaOH (50 mL). The mixture was transferred to aseparatory funnel and extracted with EtOAc (2×50 mL). The organic phasewas washed with brine (50 mL) and dried (Na₂SO₄). The crude material wassubject to flash chromatography on silica gel (10% EtOAc/hexanes then20% EtOAc/hexanes). The sample was subject to additional flashchromatography on silica gel (7.5-15% EtOAc/hexanes) to give viscous oil(0.41 g, 19%). ¹H NMR (CDCl₃) δ 7.08 (dd, J=0.9, 7.5 Hz, 1H), 7.04-6.99(m, 1H), 6.77 (d, J=8.1 Hz, 1H), 6.67-6.72 (m, 1H), 4.39-4.36 (m, 1H),3.73-3.72 (m, 6H), 3.10-3.06 (m, 2H), 2.20-2.00 (m, 2H), 1.47 (s, 9H);ESI-MS (m/z, %): 345, 343 (M+Na, 4), 322, 320 (MH⁺, 3), 267, 265 (100).

tert-Butyl3-(7-bromo-2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate

A solution of tert-butyl3-(2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate (0.41 g,1.302 mmol) in DMF (5 mL), was cooled to 0° C. and treated dropwise withN-bromosuccinimide (0.17 g, 0.977 mmol) in DMF (5 mL) for 15 minutes.The reaction was kept at 0° C. and was stirred for 1 hour at thistemperature. At this time, N-bromosuccinimide (0.023 g, 0.130 mmol) inDMF (1 mL) was added dropwise to the reaction, which was then stirredfor 1 hour at 0° C. The solution was treated again withN-bromosuccinimide (0.023 g, 0.130 mmol) in DMF (1 mL) and was stirredfor 1 hour at 0° C. The solution was diluted in water (100 mL) andextracted with EtOAc (2×100 mL). The aqueous phase was washed once withEtOAc (50 mL). The organic solutions were combined, washed with water(100 mL) and brine (50 mL), and dried (Na₂SO₄). The crude material wasfiltered through a silica pad (20% EtOAc/hexanes) and concentrated togive clear oil (0.47 g, 90%). ¹H NMR (CDCl₃) δ 7.18 (d, J=2.4 Hz, 1H),7.07 (d, J=8.7 Hz, 1H), 6.62 (d, J=8.7 Hz, 1H), 4.34-4.28 (m, 1H),3.71-3.23 (m, 6H), 3.03-3.04 (m, 2H), 2.19-1.95 (m, 2H), 1.47 (s, 9H);ESI-MS (m/z, %): 423, 421 (M+Na, 8), 401, 399 (MH⁺, 2), 343, 345 (100).

tert-Butyl3-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate

Tris(dibenzylideneacetone)dipalladium(0) (0.056 g, 0.062 mmol) in THF (3mL) was treated with tri-tert-butylphosphine 10% wt in hexanes (0.748mL, 0.247 mmol) and was stirred vigorously under argon atmosphere for 10minutes. tert-Butyl3-(7-bromo-2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate(0.49 g, 1.233 mmol) in THF (9 mL) and 1M LiHMDS in THF (3.70 mL, 3.70mmol) were added to the mixture. The reaction was sealed, placed in apreheated oil bath, and refluxed at 100° C. The solution was cooled toroom temperature, treated with 1M TBAF in THF (8 mL), and was stirredfor 40 minutes. The mixture was concentrated and treated with 1N NaOH(50 mL). The mixture was transferred to a separatory funnel, dilutedwith water (50 mL), and extracted with EtOAc (2×50 mL). The organicphase was washed with brine (50 mL) and dried (Na₂SO₄). The crudematerial was subject to flash chromatography on silica gel (25-70%EtOAc/hexanes). The collected fractions were concentrated to give aclear brown oil (0.34 g, 83%). ¹H NMR (CDCl₃) δ 6.64 (d, J=8.7 Hz, 1H),6.48 (s, 1H), 6.39 (brs, 1H), 4.08-4.02 (m, 1H), 3.70-3.07 (m, 6H),3.09-3.07 (m, 2H), 2.08-1.92 (m, 2H), 1.46 (s, 9H); ESI-MS (m/z, %):338, 336 (MH⁺, 40), 282, 280 (100).

tert-Butyl3-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate

Methyl thiophene-2-carbimidothioate hydroiodide (0.53 g, 1.874 mmol) wasadded to a mixture of tert-butyl3-(7-amino-2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate(0.31 g, 0.937 mmol) in EtOH (20 mL). The mixture was stirred under roomtemperature overnight. The mixture was quenched with saturated sodiumbicarbonate solution (50 mL). The solution was then transferred to aseparatory funnel, diluted with water (50 mL), and extracted with CH₂Cl₂(2×50 mL). The crude material was subjected to flash chromatography onsilica gel (50-90% EtOAc/hexanes). The concentrated fractions afforded ayellow oil (0.29 g, 69%). ¹H NMR (CDCl₃) δ 7.41 (dd, J=0.9, 5.1 Hz, 1H),7.38 (d, J=3.6 Hz, 1H), 7.06 (dd, J=3.9, 4.9 Hz, 1H), 6.79-6.76 (m, 2H),6.68-6.66 (m, 1H), 4.84 (brs, 2H), 4.35-4.23 (m, 1H), 3.74-3.22 (m, 6H),3.12-3.07 (m, 2H), 2.19-1.99 (m, 2H), 1.47 (s, 9H); ESI-MS (m/z, %):447, 445 (MH⁺, 100).

N-(4-(Pyrrolidin-3-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A solution of tert-butyl3-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)pyrrolidine-1-carboxylate(0.24 g, 0.554 mmol) in MeOH (5 mL) was treated with 1N HCl solution(9.98 mL, 9.98 mmol) at room temperature. The resulting mixture wasrefluxed for 30 minutes and brought to room temperature. The mixture wasfiltered and quenched with 1N NaOH (10 mL). The mixture was diluted withwater (50 mL) and extracted with CH₂Cl₂ (2×50 mL). The organic phase waswashed with brine (50 mL) and dried (Na₂SO₄). The crude material wassubject to flash chromatography on silica gel (5-20% 2M NH₃MeOH/CH₂Cl₂).The collected fractions gave compound 30 as yellow foam (0.15 g, 79%).¹H NMR (DMSO-d₆) δ 7.69 (d, J=3.0 Hz, 1H), 7.56 (dd, J=0.9, 4.9 Hz, 1H),7.07 (dd, J=3.9, 4.9 Hz, 1H), 6.79 (d, J=9.0 Hz, 1H), 6.52-6.49 (m, 2H),6.31 (brs, 2H), 4.26-4.17 (m, 1H), 3.42-3.22 (m, 2H), 3.10-2.90 (m, 4H),2.83-2.69 (m, 2H), 2.07-1.96 (m, 1H), 1.68-1.57 (m, 1H); ESI-MS (m/z,%): 347, 345 (MH⁺, 72), 278, 276 (100).

N-(4-(Pyrrolidin-3-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

N-(4-(Pyrrolidin-3-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.1264 g, 0.367 mmol) in MeOH (3 mL) was treated with 1M HCl in ether(1.835 mL, 1.835 mmol). The mixture was stirred at room temperature for5 minutes and was concentrated to give a light yellow solid (0.16 g,quantitative). ¹H NMR (DMSO-d₆) δ 11.30 (s, 1H), 9.83 (brs, 1H), 9.70(s, 1H), 9.57 (brs, 1H), 8.70 (s, 1H), 8.15-8.13 (m, 2H), 7.37-7.34 (m,1H), 7.10-7.00 (m, 3H), 4.74-4.63 (m, 1H), 3.60-3.40 (m, 5H), 3.16-3.05(m, 3H), 2.26-2.15 (m, 1H), 2.05-1.92 (m, 1H); ESI-MS (m/z, %): 347, 345(MH⁺, freebase, 61), 278, 276 (100); HRMS (C₁₇H₂₁N₄S₂, MH⁺, freebase):calculated: 345.1202, observed: 345.1195. HPLC purity: 99% by area.

Example 31 Synthesis ofN-(7-fluoro-4-(piperidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamide(31)

2-(2-Amino-5-fluoro-4-nitrophenylthio)ethanol

To a stirred suspension of 2,4-difluoro-5-nitroaniline (100 mg, 0.574mmol) and potassium carbonate (159 mg, 1.149 mmol) in DMF (1 mL) wasadded 2-mercaptoethanol (0.081 mL, 1.149 mmol). The resulting mixturewas stirred at room temperature for 2 hours. The mixture was thendiluted with ethyl acetate and washed with saturated sodium carbonate(3×). The organic phase was dried, filtered, concentrated, and thenchromatographed (1:1 EtOAc:hexanes) to give the major product. Ethylacetate was then used as the eluent to give a minor product. The majorproduct was determined to be the desired product (112 mg, 84%). ¹H NMR(DMSO-d₆) δ 7.32 (d, J=3.5 Hz, 1H), 7.27 (d, J=10.7 Hz, 1H), 4.39 (s,2H), 3.85-3.76 (m, 2H), 3.16-3.08 (m, 2H), 2.13-2.05 (m, 1H); ESI-MS(m/z, %): 233 (MH⁺, 100), 215 (14), 187 (100), 129 (65).

tert-Butyl4-(4-fluoro-2-(2-hydroxyethylthio)-5-nitrophenylamino)piperidine-1-carboxylate

To a stirred solution of 2-(2-amino-5-fluoro-4-nitrophenylthio)ethanol(434 mg, 1.869 mmol) and tert-butyl 4-oxopiperidine-1-carboxylate (372mg, 1.869 mmol) in dichloroethane (10 mL) and acetic acid (0.321 mL,5.61 mmol) was added sodium triacetoxyborohydride (594 mg, 2.80 mmol).The resulting mixture was stirred at room temperature. After 1 hour,additional tert-butyl 4-oxopiperidine-1-carboxylate (372 mg, 1.869 mmol)was added, followed by additional sodium triacetoxyborohydride (594 mg,2.80 mmol). After a further 3 hours, more tert-butyl4-oxopiperidine-1-carboxylate (372 mg, 1.869 mmol) was added followed bymore sodium triacetoxyborohydride (594 mg, 2.80 mmol). The reactionmixture was stirred overnight. The mixture was then quenched with 1 NNaOH (10 mL), water (20 mL), and saturated sodium carbonate (20 mL). Theorganic products were then extracted with dichloromethane (3×20 mL). Thecombined organics were dried, filtered, concentrated, and thenchromatographed using 1:3 ethyl acetate:hexanes on silica gel to givethe desired product (710 mg, 91%). ¹H NMR (DMSO-d₆) δ 7.45 (d, J=12.0Hz, 1H), 7.23 (d, J=6.6 Hz, 1H), 5.08 (t, J=5.4 Hz, 1H), 5.02 (d, J=8.1Hz, 1H), 3.96-3.84 (m, 2H), 3.69-3.55 (m, 5H), 2.99-2.83 (m, 2H),1.92-1.83 (m, 2H), 1.45-1.18 (m, 11H); ESI-MS (m/z, %): 360 (100), 128(30).

tert-Butyl4-(4-fluoro-2-(2-iodoethylthio)-5-nitrophenylamino)piperidine-1-carboxylate

To a stirred solution of triphenylphosphine (668 mg, 2.55 mmol) andimidazole (0.693 mL, 5.09 mmol) in tetrahydrofuran (10 mL) cooled to 0°C. was added iodine (732 mg, 2.88 mmol). The resulting mixture wasstirred at 0° C. for 5 minutes. To this mixture was then addedtert-butyl4-(4-fluoro-2-(2-hydroxyethylthio)-5-nitrophenylamino)piperidine-1-carboxylate(705 mg, 1.697 mmol) as a solution in tetrahydrofuran (5 mL), and theresulting mixture was stirred at room temperature for 20 minutes. Themixture was then diluted with ethyl acetate and washed with saturatedsodium carbonate, water, saturated sodium thiosulfate, and brine. Theorganic phase was dried, filtered, concentrated, and thenchromatographed in 1:9 ethyl acetate:hexanes to afford the desiredproduct (398 mg, 44.6%). ¹H NMR (DMSO-d₆) δ 7.53 (d, J=11.7 Hz, 1H),7.27 (d, J=6.6 Hz, 1H), 5.10 (d, J=8.1 Hz, 1H), 3.96-3.85 (m, 2H),3.64-3.52 (m, 1H), 3.50-3.41 (m, 2H), 3.39-3.32 (m, 2H), 2.99-2.81 (m,2H), 1.91-1.81 (m, 2H), 1.48-1.32 (m, 11H); ESI-MS (m.z, %): 470 (100),426 (81).

tert-Butyl4-(7-fluoro-6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate

To a stirred solution of tert-butyl4-(4-fluoro-2-(2-iodoethylthio)-5-nitrophenylamino)piperidine-1-carboxylate(394 mg, 0.750 mmol) in DMF (5 mL) was added potassium carbonate (207mg, 1.500 mmol), and the resulting mixture was stirred at 60° C. for 2hours and then at 90° C. overnight. The mixture was then cooled to roomtemperature, diluted with ethyl acetate, and washed with saturatedsodium carbonate (3×), water, and brine. The organic phase was dried,filtered, concentrated, and then chromatographed using 1:9 ethylacetate:hexanes giving the desired product (193 mg, 64.7%) as a redfoam. ¹H NMR (DMSO-d₆) δ 7.42 (d, J=6.6 Hz, 1H), 7.30 (d, J=11.7 Hz,1H), 4.10-3.95 (m, 2H), 3.91-3.79 (m, 1H), 3.41-3.36 (m, 2H), 3.13-3.09(m, 2H), 2.99-2.78 (m, 2H), 1.73-1.63 (m, 2H), 1.63-1.51 (m, 2H), 1.41(s, 9H); ESI-MS (m/z, %): 420 (MNa⁺, 52), 398 (MH⁺, 35), 342 (100), 298(64).

tert-Butyl4-(6-amino-7-fluoro-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate

To a stirred solution of tert-butyl4-(7-fluoro-6-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate(190 mg, 0.478 mmol) in tetrahydrofuran (3.00 mL) was added Raney-Nickel(28.1 mg, 0.478 mmol) as a suspension in methanol (3 mL). To thismixture was then added hydrazine hydrate (0.233 mL, 4.78 mmol), and theresulting mixture was stirred at 60° C. for 8 minutes. During this time,the reaction mixture turns light red. The mixture was then diluted withethyl acetate and washed with saturated sodium carbonate (2×), water(2×), and brine. The organic phase was dried, filtered, andconcentrated, giving a red oil (172 mg, 98%). ¹H NMR (DMSO-d₆) δ 6.61(d, J=11.1 Hz, 1H), 6.34 (d, J=8.4 Hz, 1H), 4.76 (s, 2H), 4.11-4.01 (m,2H), 3.62-3.51 (m, 1H), 3.29-3.25 (m, 2H), 2.93-2.88 (m, 2H), 2.87-2.69(m, 2H), 1.71-1.61 (m, 2H), 1.59-1.48 (m, 2H), 1.40 (m, 9H); ESI-MS(m/z, %): 368 (MH⁺, 25), 312 (100).

tert-Butyl4-(7-fluoro-6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate

To a stirred solution of tert-butyl4-(6-amino-7-fluoro-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate(168 mg, 0.457 mmol) in ethanol (5 mL) and triethylamine (0.643 mL, 4.57mmol) was added methyl thiophene-2-carbimidate hydrochloride (325 mg,1.829 mmol). The resulting mixture was stirred at 65° C. over theweekend. The mixture was then cooled to room temperature, diluted withethyl acetate, and washed with saturated sodium carbonate (3×). Theorganic phase was dried, filtered, and concentrated, and chromatographedin 1:9 ethyl acetate:hexanes giving a yellow oil (18 mg, 8.26%); ¹H NMR(MeOD-d₄) δ 7.65-7.61 (m, 1H), 7.59-7.54 (m, 1H), 7.14-7.08 (m, 1H),6.79 (d, J=10.5 Hz, 1H), 6.49 (d, J=7.5 Hz, 1H), 4.22-4.12 (m, 2H),3.80-3.67 (m, 1H), 3.43-3.38 (m, 2H), 3.05-3.01 (m, 2H), 2.96-2.79 (m,2H), 1.83-1.73 (m, 2H), 1.72-1.57 (m, 2H), 1.46 (s, 9H); ESI-MS (m/z,%): 477 (MH⁺, 100).

N-(7-Fluoro-4-(piperidin-4-yl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-6-yl)thiophene-2-carboximidamidedihydrochloride

To a stirred solution of tert-butyl4-(7-fluoro-6-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)piperidine-1-carboxylate(16 mg, 0.034 mmol) in methanol (1 mL) was added 1 drop of concentratedhydrochloric acid. The resulting mixture was heated at 70° C. for 1hour. The mixture was then cooled to room temperature and diluted withdiethyl ether (4 mL). The resulting pale precipitate was collected viavacuum filtration, giving the desired dihydrochloride salt product 31 (8mg, 53.0%). ¹H NMR (DMSO-d₆) δ 11.35 (s, 1H), 9.92 (s, 1H), 8.96 (s,1H), 8.57-8.51 (m, 2H), 8.25-8.10 (m, 2H), 7.40 (s, 1H), 7.24-7.15 (m,1H), 7.05-6.99 (m, 1H), 3.96-3.83 (m, 1H), 3.45-3.20 (m, 2H), 3.18-3.09(m, 2H), 3.09-2.91 (m, 2H), 2.02-1.87 (m, 2H), 1.87-1.75 (m, 2H); ¹H NMR(DMSO-d₆+D₂O) δ 8.16-8.09 (m, 1H), 8.08-8.01 (m, 1H), 7.39-7.33 (m, 1H),7.14 (d, J=10.5 Hz, 1H), 6.95-6.88 (m, 1H), 3.91-3.80 (m, 1H), 3.39-3.29(m, 4H), 3.11-3.05 (m, 2H), 3.05-2.91 (m, 2H), 1.90-1.78 (m, 4H); ESI-MS(m/z, %): 377 (MH⁺, free base, 50), 294 (100); ESI-HRMS (C₁₈H₂₂N₄FS₂,MH⁺, free base), calculated: 377.1264, observed: 377.1263.

Example 32 Synthesis ofN-(4-(2-(ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(32)

2-Chloro-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

Prepared according to the reported procedure in Example 18.

2-(Ethylamino)-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone

A solution of2-chloro-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone (1.5 g,5.50 mmol) in dioxane (15 mL) and triethylamine (1.533 mL, 11.00 mmol)was treated with a solution of ethanamine hydrochloride (2.243 g, 27.5mmol) in water (7.5 mL). The reaction was then stirred at roomtemperature overnight (16 hours). At this time, the reaction mixture wasdiluted with water (50 mL) and extracted into dichloromethane (3×30 mL).The combined organic layer was dried (Na₂SO₄), filtered, andconcentrated. The residue was subjected to column chromatography onsilica gel (1:1 ethyl acetate:hexanes then 5:95 (2M NH₃ in MeOH):CH₂Cl₂)to give a brown solid (0.56 g, 36.2%). ¹H-NMR (DMSO-d₆) δ 8.10 (d, J=5.1Hz, 1H), 7.92 (dd, J=2.4, 9.0 Hz, 1H), 7.70 (d, J=9.0 Hz, 1H), 3.94 (t,J=5.1 Hz, 2H), 3.52 (s, 2H), 3.33-3.28 (m, 2H), 2.55-2.52 (m, 2H), 0.98(t, J=6.9 Hz, 3H); ESI-MS (m/z, %): 282 (MH⁺, 100%).

tert-Butylethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate

A solution of2-(ethylamino)-1-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethanone(0.56 g, 1.991 mmol) in anhydrous dioxane (10 mL) and triethylamine(0.555 mL, 3.98 mmol) was treated with di-tert-butyl dicarbonate (0.478g, 2.190 mmol) and stirred at room temperature for half an hour. Thereaction was then diluted with ethyl acetate (50 mL) and washed withwater (50 mL) and brine (3×15 mL). The organic layer was dried (Na₂SO₄),filtered, and concentrated to give a brown syrup (0.803 g, 100%). ¹H-NMR(DMSO-d₆) δ 8.13-8.11 (m, 1H), 7.97-7.91 (m, 1H), 7.71 (d, J=9.0 Hz,1H), 4.18-4.16 (m, 2H), 3.95-3.92 (m, 2H), 3.28-3.19 (m, 4H), 1.38, 1.29(2×s, 9H), 1.05-1.02 (m, 3H); ESI-MS (m/z, %): 404 (M+Na, 42%), 382(MH⁺, 17%), 282 (100%).

tert-Butylethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

A solution of tert-butylethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)-2-oxoethyl)carbamate(0.8 g, 2.097 mmol) in anhydrous tetrahydrofuran (5 mL) was treated withborane-THF complex (1M in THF; 6.29 mL, 6.29 mmol) and stirred at roomtemperature for 3 hours. The reaction was quenched dropwise withmethanol (10 mL) and then concentrated. The crude residue was dilutedwith methanol (25 mL) and refluxed for 10 minutes. The mixture was thenconcentrated, and the residue was subjected to column chromatography onsilica gel (1:9-3:7 EtOAc:CH₂Cl₂) to give a yellow syrup (0.493 g, 64%).¹H-NMR (DMSO-d₆) δ 7.85-7.78 (m, 2H), 6.94-6.85 (m, 1H), 3.76-3.79 (m,2H), 3.62-3.57 (m, 2H), 3.37-3.33 (m, 2H), 3.22-3.19 (m, 2H), 3.08-3.04(m, 2H), 1.35 and 1.30 (2×brs, 9H), 1.03 (t, J=6.9 Hz, 3H). ESI-MS (m/z,%): 390 (M+Na, 67%), 368 (MH⁺, 17%), 312 (83%), 268 (100%).

tert-Butylethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate

A suspension of palladium on carbon (10% wt; 0.142 g, 0.133 mmol), andtert-butylethyl(2-(7-nitro-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate (0.49g, 1.333 mmol) in dry ethanol (15 mL) was stirred under hydrogen gas(balloon pressure) for 3 hours. The reaction was then placed underargon, methyl thiophene-2-carbimidothioate hydroiodide (0.761 g, 2.67mmol) was added, and the mixture stirred at room temperature overnight(16 hours). The mixture was poured over Celite, and the Celite wasrinsed with methanol. The filtrate was concentrated and subjected tocolumn chromatography on silica gel (1:1 EtOAc:hexanes then 5:95 (2M NH₃in MeOH):CH₂Cl₂) to give a solid (0.4 g, 67.2%). ¹H-NMR (DMSO-d₆) δ 7.69(brd, J=3.3 Hz, 1H), 7.57 (dd, J=1.2, 5.2 Hz, 1H), 7.07 (dd, J=3.6, 4.8Hz, 1H), 6.81-6.74 (m, 1H), 6.49-6.46 (m, 2H), 6.31 (brs, 2H), 3.59-3.54(m, 2H), 3.36-3.31 (overlap with solvent peak m, 4H), 3.23-3.20 (m, 2H),3.03-3.00 (m, 2H), 1.41 (s, 9H), 1.04 (t, J=6.9 Hz, 3H); ESI-MS (m/z,%): 447 (MH⁺, 100%).

N-(4-(2-(Ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A solution of tert-butylethyl(2-(7-(thiophene-2-carboximidamido)-2H-benzo[b][1,4]thiazin-4(3H)-yl)ethyl)carbamate(0.4 g, 0.896 mmol) in methanol (10 mL) was treated with 3 N HCl (3.88mL, 11.64 mmol) and stirred at reflux for 1.5 hours. The mixture wascooled to room temperature, diluted with water (20 mL), and basifiedwith 1N NaOH. The product was extracted into dichloromethane (2×25 mL).The combined organic layer was dried (Na₂SO₄) and concentrated. Thecrude material was subjected to column chromatography on silica gel(5:95 (2M NH₃ in MeOH):CH₂Cl₂) to give a yellow solid (0.142 g, 45.8%).¹H-NMR (DMSO-d₆) δ 7.69 (dd, J=0.9, 3.6 Hz, 1H), 7.56 (d, J=5.1 Hz, 1H),7.07 (dd, J=3.6, 4.8 Hz, 1H), 6.72 (d, J=8.7 Hz, 1H), 6.49 (dd, J=2.1,8.4 Hz, 1H), 6.45 (d, J=2.1 Hz, 1H), 6.31 (brs, 2H), 3.55-3.51 (m, 2H),3.30-3.24 (m, 2H), 3.04-3.00 (m, 2H), 2.62 (t, J=6.9 Hz, 2H), 2.56 (q,J=7.2 Hz, 2H), 1.01 (t, J=7.2 Hz, 3H); ESI-MS (m/z, %): 347 (MH⁺, 75%),276 (100%); HPLC purity: 99.5% by area.

N-(4-(2-(Ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamidedihydrochloride

A solution ofN-(4-(2-(ethylamino)ethyl)-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(0.142 g, 0.410 mmol) in dry methanol (5 mL) was treated withhydrochloric acid (1M in ether; 2.049 mL, 2.049 mmol) and stirred atroom temperature for 1 hour. The reaction was then concentrated to givea yellow solid. ¹H-NMR (DMSO-d₆) δ 11.22 (brs, 1H) 9.68 (brs, 1H), 9.32(brs, 2H), 8.69 (brs, 1H), 8.15-8.11 (m, 2H), 7.36 (pseudo t, J=7.2 Hz,1H), 7.06-6.99 (m, 3H), 3.70-3.60 (overlap with solvent peak, m, 4H),3.12-3.02 (m, 4H), 3.02-2.93 (m, 2H), 1.23 (t, J=7.2 Hz, 3H).

Example 33 Synthesis ofN-(4-(2-(dimethylamino)ethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide(33)

4-(2-(Dimethylamino)ethyl)-7-nitro-2H-benzo[b][1,4]thiazin-3(4H)-one

Prepared according to the reported procedure in Example 8.

N-(4-(2-(Dimethylamino)ethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazin-7-yl)thiophene-2-carboximidamide

A suspension of4-(2-(dimethylamino)ethyl)-7-nitro-2H-benzo[b][1,4]thiazin-3(4H)-one(0.3 g, 1.066 mmol) and palladium on carbon (10% wt, 0.113 g, 0.107mmol) in dry ethanol (10 mL) was stirred under a hydrogen atmosphere(balloon pressure) for 2 hours. The mixture was passed over a pad ofCelite, and the Celite was rinsed with ethanol. The filtrate was treatedwith methyl thiophene-2-carbimidothioate hydroiodide (0.608 g, 2.133mmol), and this reaction mixture was stirred at room temperature for 3days. The solvent was evaporated, and the residue was partitionedbetween dichloromethane (50 mL) and saturated sodium bicarbonatesolution (25 mL), stirring for half an hour. The mixture was thentransferred to a separatory funnel, and the aqueous layer was removed.The organic layer was dried (Na₂SO₄) and concentrated. The residue wassubjected to column chromatography on silica gel (2.5:97.5 (2M NH₃ inMeOH):CH₂Cl₂ then 5:95 (2M NH₃ in MeOH):CH₂Cl₂ then 7.5:92.5 (2M NH₃ inMeOH):CH₂Cl₂) to give a yellow solid (0.117 g, 30.4%). ¹H-NMR (DMSO-d₆)δ 7.75 (brd, J=3.0 Hz, 1H), 7.61 (dd, J=0.9, 5.1 Hz, 1H), 7.28 (d, J=8.7Hz, 1H), 7.09 (dd, J=3.6, 4.8 Hz, 1H), 6.85 (brd, J=1.8 Hz, 1H), 6.79(dd, J=1.5, 8.4 Hz, 1H), 6.58 (brs, 2H), 4.00 (t, 6.9 Hz, 2H), 3.44 (s,2H), 2.41 (t, J=9.0 Hz, 2H), 2.19 (s, 6H).

Example 34 nNOS (Human), eNOS (Human) and iNOS (Human) Enzyme Assay

The human iNOS assay was carried by either one of the two protocols asdescribed herein:

Protocol 1

Recombinant human inducible NOS (iNOS) was produced inBaculovirus-infected Sf9 cells (ALEXIS). In a radiometric method, NOsynthase activity was determined by measuring the conversion of[³H]L-arginine to [³H]L-citrulline. To measure iNOS, 10 μL of enzyme wasadded to 100 μL of 100 mM HEPES, pH=7.4, containing 1 mM CaCl₂, 1 mMEDTA, 1 mM dithiothreitol, 1 μM FMN, 1 μM FAD, 10 μMtetrahydrobiopterin, 120 μM NADPH, and 100 nM CaM.

To measure enzyme inhibition, a 15 μL solution of a test substance wasadded to the enzyme assay solution, followed by a pre-incubation time of15 min at RT. The reaction was initiated by addition of 20 μL L-argininecontaining 0.25 μCi of [³H] arginine/mL and 24 μM L-arginine. The totalvolume of the reaction mixture was 150 μL in every well. The reactionswere carried out at 37° C. for 45 minutes, The reaction was stopped byadding 20 μL of ice-cold buffer containing 100 mM HEPES, 3 mM EGTA, 3 mMEDTA, pH=5.5. [³H]L-citrulline was separated by DOWEX (ion-exchangeresin DOWEX 50 W×8-400, SIGMA) and the DOWEX was removed by spinning at12,000 g for 10 min in the centrifuge. An aliquot (70 μL) of thesupernatant was added to 100 μL of scintillation fluid, and the sampleswere counted in a liquid scintillation counter (1450 Microbeta Jet,Wallac). Specific NOS activity was reported as the difference betweenthe activity recovered from the test solution and that observed in acontrol sample containing 240 mM of the inhibitor L-NMMA. All assayswere performed at least in duplicate. Standard deviations are 10% orless. These results show the selectivity of the compounds of theinvention for nNOS inhibition. Results for exemplary compounds of theinvention are also shown in Table 3.

Protocol 2:

Reagents and Materials: Enzyme Nitric oxide synthase (inducible, humanrecombinant) iNOS II, Cat. No. ALX-201-060, Axxora LLC, CA 92121, USAL-NMMA N^(G)-monomethyl-L-arginine dihydrochloride, Cat # M7033, SigmaAldrich Reaction Buffer 50 mM Tris-HCl (pH 7.4), Cat. No. 93313,Sigma-Aldrich Co., St. Louis, MO 6 uM tetrahydrobiopterindihydrochloride (BH₄), Cat. No. T4425, Sigma 2 uM flavin adeninedinucleotide (FAD), Cat. No. F6625, Sigma 2 uM flavin adeninemononucleotide (FMN), Cat. No. F8399, Sigma Stop Buffer 50 mMN-2-hydroxyethylpiperazine-N′-2- ethanesulfonic acid (HEPES) (pH 5.5),H7523, Sigma and 5 mM Ethylene diamine tetraacetic acid (EDTA), Cat. No.EDS, Sigma NADPH 12 mM prepared in 10 mM Tris-HCl (pH 7.4), Cat. No.N7505, Sigma Calmodulin 1 μM, Cat. No. P2277, Sigma [³H]-L-Arginine 1μCi/reaction, 40-70 Ci/mmol, Cat. No. TRK- 698, Amersham BiosciencesL-Arginine 2.5 μM (final assay concentration), Cat. No. A5131, SigmaEquilibrated Resin AG-50W X8 Resin in HEPES buffer (pH 5.5), Cat. No.1421441, Bio-Rad Laboratories Ltd. Spin Cups Cat. No. C8163, FisherScientific Microbeta Wallac Microbeta Trilux, Perkin Elmer LiquidScintillation Optiphase Supermix, Cat. No. 1200-439, Perkin Fluid ElmerIncubator Lab-Line Enviro Shaker Microcentrifuge Mikro 20

Procedure

Primary stock solutions of each test compound were prepared in distilledwater on the day of study to obtain a final concentration of 6 mM. Forthe determination of IC₅₀ values, 11 test compound concentrations wereprepared as 3-fold serial dilutions. Test compounds were tested in theiNOS assay at a concentration of 1000 μM to 0.0169 μM. The vehicle ofthe test compound or inhibitor, distilled water, was used as blankcontrol. For non-specific activity, 100 μM L-NMMA was run in parallel asa control.

All incubations were performed in duplicate

The reaction mixture was prepared on ice by the addition of thefollowing components to the appropriate well of a 1.0 mL 96-wellpolypropylene plate:

10 μL of test compound, reference inhibitor, or control (vehicle orL-NMMA) solution;

25 μL of Reaction Buffer (50 mM Tris-HCl, 6 μM BH₄, 2 μM FMN, 2 μM FAD);

5 μL of 10 mM NADPH solution;

5 μL of distilled water;

5 μL of 1 μM Calmodulin; and

5 μL of 0.0024 μg/μL iNOS.

The reaction mixture was pre-incubated at room temperature for 15minutes. Following this pre-incubation, the reaction was initiated bythe addition of 5 μL of the substrate L-arginine (1 μCi of[³H]-L-Arginine+30 μM of unlabeled L-Arginine) to the reaction mixture.The total reaction volume was 60 μL. The assay plate containing thereaction mixtures was sealed and incubated at 37° C. for 30 minutes. Atthe end of this incubation period, 400 μL of ice-cold Stop Buffer (theEDTA in the Stop Buffer chelates all of the available calcium) was addedto each well to stop the reaction. 100 μL of equilibrated resin pH 5.5was added to each of the assay wells. The reaction samples were vortexedand transferred to spin cups, after-which they were centrifuged at13,000 rpm for 30 sec. The spin cups were removed from the holder and 20eluate (containing the unbound L-citrulline) were added to theappropriate wells of a 96-well liquid scintillation plate. 175 μL ofscintillation fluid were also added to each well. The plate was sealed,and radioactivity was quantitated using a Microbeta liquid scintillationcounter.

Data were analyzed using a Sigmoidal dose-response (variable slope)curve to determine the IC₅₀ value of the test compound.Y=Bottom+(Top−Bottom)/(1+10^((Log IC₅₀ −X)*Hill Slope))

X is the logarithm of test compound or inhibitor concentration

Y is the amount of L-citrulline formation (pmol)

Bottom refers to the lowest Y value and Top refers to the highest Yvalue.

This is identical the “four parameter logistic equation”

The slope factor (also called Hill slope) describes the steepness of acurve. A standard competitive binding curve that follows the law of massaction has a slope of −1.0. If the slope is shallower, the slope factorwill be a negative fraction (for example, −0.85 or −0.60).

Human nNOS and eNOS Protocol:

Reagents and Materials Enzymes: Nitric oxide synthase (neuronal, humanrecombinant) nNOS I, Cat. No. ALX-201-068, Axxora LLC, CA 92121, USANitric oxide synthase (endothelial, human recombinant) eNOS III, Cat.No. ALX-201-070, Axxora LLC L-NMMA: N^(G)-monomethyl-L-arginine 1/04/05,Cat # A17933, Novabiochem L-NAME: N^(G)-Nitro-L-arginine methyl esterCat # N5751, Aldrich 2× Reaction Buffer: 50 mM Tris-HCl (pH 7.4), Cat.No. 93313, Sigma-Aldrich Co., St. Louis, MO 6 μM tetrahydrobiopterin(BH₄), Cat. No. T4425, Sigma 2 μM flavin adenine dinucleotide (FAD),Cat. No. F6625, Sigma 2 μM flavin adenine mononucleotide (FMN), Cat. No.F8399, Sigma Stop Buffer: 50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 5.5), H7523, Sigma and 5 mMEthylenediaminetetraacetic acid (EDTA), Cat. No. EDS, Sigma NADPH: 10 mMfreshly prepared on day of assay, Cat. No. N7505, Sigma CalciumChloride: 6 mM, Cat. No. 21107, Sigma Calmodulin: 1 mM, Cat. No. P2277,Sigma [³H]-L -Arginine: 1 μCi/reaction, 40-70 Ci/mmol, Cat. No. TRK-698, Amersham Biosciences L-Arginine: 2.5 μM (final assayconcentration), Cat. No. A5131, Sigma Equilibrated Resin: AG-50W X8Resin in HEPES buffer (pH 5.5), Cat. No. 1421441, Bio-Rad LaboratoriesLtd. Spin Cups & Holder: Cat. No. C8163, Fisher Scientific LiquidScintillation Tri-Carb 2000CA/LL, Canberra Packard Counter: CanadaLiquid Scintillation Cat. No. 6012239, Ultima Gold, Perkin-Elmer Fluid:Life and Analytical Sciences, MA CO₂ Incubator: Lab-Line Enviro ShakerMicrocentrifuge: Mikro 20 Vortex Mixer: Mini Vortex mixer, IKAProcedure for Human nNOS and eNOS

Primary stock solutions of test compounds at a concentration of 6 mMwere prepared from the 2 to 5 mg powder. The primary stock solutions ofeach test compound were prepared freshly in distilled water on the dayof study to obtain a final concentration of 6 mM. For determination ofIC₅₀ values, 12 test compound concentrations were prepared as 3-foldserial dilutions. Concentration range of test compound utilized for nNOSwere 0.001 to 300 μM and for eNOS were 0.003 to 1000 μM. The vehicle ofthe test compound or inhibitor was used as blank control. Fornon-specific activity, 100 μM L-NMMA was used. Runs using the IC₅₀concentration of L-NAME were done in parallel as controls.

All incubations are performed in duplicate

The reaction mixture was prepared on ice by adding the followingcomponents with a micropipette to a polypropylene microcentrifuge tube:

-   -   10 μL of test compound, inhibitor or control (vehicle or L-NMMA)        solution;    -   25 μL of Reaction Buffer {25 mM Tris-HCl, 0.6 μM BH₄, 0.2 μM        FMN, 0.2 μM FAD};    -   5 μL of 10 mM NADPH solution {1 mM} (freshly prepared in 10 mM        Tris-HCl (pH 7.4);    -   5 μL of 6 mM CaCl_(2 {)600 W};    -   5 μL of 1 mM Calmodulin {100 W}; and    -   5 μL of 0.02 μg/μL nNOS or 0.12 μg/μL eNOS.

The above reaction mixture was pre-incubated at room temperature for 15mins.

The reaction was started by the addition of the substrate (in 5 μLcontaining 1 μCi of [³H]-L-Arginine+2.5 μM of unlabeled L-Arginine) tothe reaction mixture. Total reaction volume is 60 μL.

The reaction was mixed using a vortex mixer and incubate the abovereaction mixture at 37° C. in an incubator for 30 minutes.

To stop the reaction, 400 μL of ice-cold Stop Buffer was added at theend of the incubation period.

The reaction was then mixed using a vortex mixer, and the reactionsamples were transferred to spin cups, which were centrifuged using amicrocentrifuge, at 13,000 rpm for 30 sec. at room temperature.

The spin cups were removed from the holder, 450 μL of eluate (containingthe unbound L-citrulline) were transferred to scintillation vials. 3 mLof scintillation fluid were added and the radioactivity was quantifiedin a liquid scintillation counter.

Data was analyzed using a Sigmoidal dose-response (variable slope) curveto determine the IC₅₀ value of the test compound.Y=Bottom+(Top−Bottom)/(1+10^((Log IC₅₀ −X)*Hill Slope))

X is the logarithm of test compound or inhibitor concentration

Y is the amount of L-citrulline formation (pmol)

Bottom refers to the lowest Y value and Top refers to the highest Yvalue.

This is identical to the “four parameter logistic equation.”

The slope factor (also called Hill slope) describes the steepness of acurve. A standard competitive binding curve that follows the law of massaction has a slope of −1.0. If the slope is shallower, the slope factorwill be a negative fraction (for example, −0.85 or −0.60). Results forexemplary compounds of the invention are also shown in Table 3.

TABLE 3 Selective inhibition of human NOS by Compounds (1)-(33) HumannNOS Human eNOS Human iNOS Selectivity Compound IC50, μM IC50, μM IC50,μM eNOS/nNOS 1 2.08 87.2 >100 41.9 2 0.43 10.9 10.6 25.3 3 0.85 13.8 1.516.2 4 0.41 25.1 2.7 61.2 5 1.84 47 >100 25.5 6 0.2 41.7 10.9 208.5 71.06 10.4 >100 9.8 8 0.06 33.5 70.6 531 9 0.20 36.5 7.25 182.5 10 0.083.82 3.74 47.7 11 0.24 24.4 117 101.6 12 0.12 19.1 62.9 159.1 13 0.199.92 33.1 52.2 14 0.14 11.5 172 82.1 15 0.15 20.1 40.7 134 16 0.034 7.058.7 205 17 1.16 10.9 241 9.3 18 0.66 12.6 341 19 19 0.14 94.7 136 67620 0.16 46.4 268 290 21 0.14 10.0 49.6 71.4 22 0.014 12.1 115 864 230.028 18.6 172 664 24 0.05 4.45 82.3 89 25 0.20 18.1 43.6 90.5 26 0.158.48 60.1 56.5 27 0.13 34.9 66.5 268.4 28 0.08 8.58 348 107.2 29 0.1143.6 76.8 396 30 NA NA NA NA 31 NA NA NA NA 32 NA NA NA NA 33 NA NA NANA NA = Data not available

Example 35 Efficacy in Models Predictive of Neuropathic Pain States

Several animal models of neuropathic pain have been described thatinvolve nerve injury including chronic constriction injury, including:CCI or Bennett model (see, for example, Bennett and Xie, Pain, 33:87-107, 1988; Vissers et al. Pharmacol. Biochem. Behav. 84: 479-486,2006), Spared Nerve Ligation (SNL or Chung model; see, for example, Kimet al. Neurosci. Lett. 199: 158-60, 1995), and the Seltzer model(Seltzer et al. Pain 43: 205-18, 1990). These models show goodcorrelation between drugs with proven activity in humans andpharmacological activity in these models (Kontinen and Meert, In:Dostrovsky J., Carr D. B., Kotzenburg M., editors. 10^(th) WorldCongress on Pain, 2003, 24. San Diego, Calif.: IASP Press, 489-98). Ingeneral, these animal models show changes in behavorial responses suchas decreased mechanical thresholds, mechanical hyperalgesia, chemicalhyperactivity, thermal hyperalgesia and cold allodynia (Dowdell et al.Pharmacol. Biochem. Behavior 80: 93-108, 2005).

The efficacy of the compounds of the invention for the treatment ofneuropathic pain was assessed using standard animal models predictive ofanti-hyperalgesic and anti-allodynic activity.

(a) Chung Model of Injury-Induced Neuropathic-Like Pain

The experimental designs for the Chung Spinal Nerve Ligation SNL Modelassay for neuropathic pain are depicted in FIGS. 1 and 2. Nerve ligationinjury was performed according to the method described by Kim and Chung(Kim and Chung, Pain 50:355-363, 1992). This technique produces signs ofneuropathic dysesthesias, including tactile allodynia, thermalhyperalgesia, and guarding of the affected paw. Rats were anesthetizedwith halothane, and the vertebrae over the L4 to S2 region were exposed.The L5 and L6 spinal nerves were exposed, carefully isolated, andtightly ligated with 4-0 silk suture distal to the DRG. After ensuringhomeostatic stability, the wounds were sutured, and the animals allowedto recover in individual cages. Sham-operated rats were prepared in anidentical fashion except that the L5/L6 spinal nerves were not ligated.Any rats exhibiting signs of motor deficiency were euthanized. After aperiod of recovery following the surgical intervention, rats showenhanced sensitivity to painful and normally non-painful stimuli.

After a standard dosing (e.g., 3 mg/kg) injected i.p. according to thepublished procedure, there is a clear effect of Compound (8) on reversalof thermal hyperalgesia (FIGS. 3 and 4).

(b) Dural Inflammation as a Rat Model of Migraine

It has been demonstrated that application of an inflammatory soup (IS)or chemical stimulus to the top of the dura in rats elicits thedevelopment of central sensitization and the appearance of mechanicalallodynia in the facial areas as well as the hindpaws of the animals.These features mimic the characteristic often seen in migraineurs duringa headache, especially when treatment is delayed, and the development ofcentral sensitization has occurred (Burstein et al., Ann. Neurol.47:614-624, 2000; Burstein et al., Brain, 123:1703-1709, 2000). Compound(8) was tested in a dural inflammation model of migraine according topreviously described procedures (WO 2007/120830 or as described inUS-2008-0031822-A1). Baseline paw withdrawal thresholds were recordedapproximately 30 min before the IS administration by application ofcalibrated von Frey filaments to the hindpaws to measure tactileallodynia. Approximately 15 minutes pre-IS administration, the testcompound in water was administered by oral gavage (1 mL/kg) at a dose of3 mg/kg per os (p.o.). Measurements of tactile allodynia were recordedat 1 hour intervals up to 6 hours post IS administration. Theantiallodynic effect of compound 8 (3 mg/kg p.o.) is shown in FIG. 5.

(c) Carrageenan-Induced Inflammation as a Model of Primary and SecondaryPain

Inflammation was induced by injecting 0.1 mL of a 3% carrageenansuspension s.c. into the supplanter aspect of a hindpaw of lightlyanesthetized rats. Typically, pronounced inflammation and edema wereobvious within three hours of the injection. The carrageenan model is amodel of inflammatory pain with two components: the first occurringimmediately after injection provides information on the acutenociceptive response to a painful stimulus (in this case, the chemicalcarrageenan). There is also a second, neurogenic component that developsseveral hours later and reflects the type of neuronal activity due tothe hyperalgesic and allodynic components as found in neuropathic pain.Considerable evidence has shown that NO and its enzymes are involved inthe central mechanisms of inflammatory hyperalgesia at the spinal cordlevel and that peripheral inflammation can upregulate nNOS expressionand induce iNOS expression in the spinal cord (Guhring et al., J.Neurosci. 20:6714-6720, 2000; Wu et al., Exp. Brain Res. 118:457-465,1998; and Wu et al., Pain 94:47-58, 2001). Furthermore, iNOS knockoutstudies in animals reveals that nNOS is essential in the development andmaintenance of the secondary components of carrageenan inflammatory painwhile iNOS is sufficient, but not essential, in the late phase ofthermal hyperalgesia (Tao et al., Neuroscience 120:847-854, 2003). FIG.6 shows the reversal of mechanical allodynia in the ipsilaterial paw ofrats after carrageenan injection and administration of Compound (8).FIG. 7 shows the reversal of thermal hyperalgesia (late phase) in theipsilateral paw of rats by Compound (8) after i.p. dosing as measured bythe paw withdrawal latency to a thermal stimulus.

Thus, a compound of Formula (I) with activity at nNOS and iNOS is usefulfor treating both migraine, inflammatory, and neuropathic type pains andother pain states wherein a component of central neuronal sensitizationexists.

Other Embodiments

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present application is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

Other embodiments are in the claims.

What is claimed is:
 1. A method of treating a condition selected fromthe group consisting of headache, neuropathic pain, chronic inflammatorypain, visceral pain, neuroinflammation, medication-induced hyperalgesiaand/or allodynia, acute pain, chronic pain, and bone cancer pain in amammal, wherein said method comprises administering an effective amountof a compound

wherein, Q is S—(CHR⁶)₁; R¹ and each R⁶ is, independently, H, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₁₋₄ alkaryl, optionallysubstituted C₁₋₄ alkheterocyclyl, optionally substituted C₂₋₉heterocyclyl, optionally substituted C₃₋₈ cycloalkyl, optionallysubstituted C₁₋₄ alkcycloalkyl, or —(CR^(1A)R^(1B))_(n)NR^(1C)R^(1D);R^(1A) and R^(1B) are, independently, H, hydroxy, halo, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₁₋₆ alkoxy, optionallysubstituted C₁₋₄ alkcycloalkyl, optionally substituted C₁₋₄ alkaryl,optionally substituted C₁₋₄ alkheterocyclyl, optionally substituted C₁₋₄alkheteroaryl, optionally substituted C₃₋₈ cycloalkyl, or optionallysubstituted C₂₋₉ heterocyclyl, or R^(1A) and R^(1B) combine to form ═O;R^(1C) and R^(1D) are, independently, H, optionally substituted C₁₋₆alkyl, optionally substituted C₁₋₆ alkoxy, optionally substituted C₁₋₄alkcycloalkyl, optionally substituted C₁₋₄ alkaryl, optionallysubstituted C₁₋₄ alkheterocyclyl, optionally substituted C₁₋₄alkheteroaryl, optionally substituted C₃₋₈ cycloalkyl, optionallysubstituted C₂₋₉ heterocyclyl, or an N-protecting group selected fromthe group consisting of formyl, acetyl, propionyl, pivaloyl,t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, alaninyl, leucinyl,phenylalaninyl, benzenesulfonyl, p-toluenesulfonyl, benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, benzyl,triphenylmethyl, benzyloxymethyl, and trimethylsilyl, or R^(1C) andR^(1D) combine to form an optionally substituted C₂₋₉ heterocyclyl; n isan integer between 1-6; each of R² and R³ is, independently, H, hal,optionally substituted C₁₋₆ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₁₋₆ alkaryl, optionally substituted C₂₋₉heterocyclyl, hydroxy, optionally substituted C₁₋₆ alkoxy, optionallysubstituted C₁₋₆ thioalkoxy, (CH₂)_(r2)NHC(NH)R^(2A), or(CH₂)_(r2)NHC(S)NHR^(2B), or optionally substituted C₁₋₄alkheterocyclyl, wherein r2 is an integer from 0 to 2, R^(2A) isoptionally substituted C₁₋₆ alkyl, optionally substituted C₆₋₁₀ aryl,optionally substituted C₁₋₄ alkaryl, optionally substituted C₂₋₉heterocyclyl, optionally substituted C₁₋₄ alkheterocyclyl, optionallysubstituted C₁₋₆ thioalkoxy, optionally substituted C₁₋₄ thioalkaryl,optionally substituted aryloyl, optionally substituted C₁₋₄thioalkheterocyclyl, or; and R^(2B) is optionally substituted C₁₋₄alkaryl, optionally substituted C₂₋₉ heterocyclyl, substituted C₁₋₄alkheterocyclyl, optionally substituted C₁₋₆ thioalkoxy, optionallysubstituted C₁₋₄ thioalkaryl, optionally substituted aryloyl, optionallysubstituted C₁₋₄ thioalkheterocyclyl, or optionally substituted amino;and each of R⁴ and R⁵ is independently H, hal, (CH₂)_(r2)NHC(NH)R^(2A),or (CH₂)_(r2)NHC(S)NHR^(2B); wherein Y¹ and Y² are each H, or Y¹ and Y²together are ═O, or Y¹ and Y² are independently H, optionallysubstituted C₁₋₆ alkyl, optionally substituted C₆₋₁₀ aryl, optionallysubstituted C₁₋₆ alkaryl, optionally substituted C₂₋₉ heterocyclyl,hydroxy, optionally substituted C₁₋₆ alkoxy, optionally substituted C₁₋₆thioalkoxy, or optionally substituted C₁₋₄ alkheterocyclyl; wherein oneand only one of R², R³, R⁴, and R⁵ is (CH₂)_(r2)NHC(NH)R^(2A) or(CH₂)_(r2)NHC(S)NHR^(2B); or a pharmaceutically acceptable salt thereofto said mammal.
 2. The method of claim 1, wherein said mammal is ahuman.
 3. The method of claim 1, wherein said headache is migraineheadache (with or without aura), chronic tension type headache (CTTH),migraine with allodynia, medication overuse headache, cluster headache,chronic headache, or transformed migraine.
 4. The method of claim 1,wherein said headache is a headache with central sensitization.
 5. Themethod of claim 1, wherein said chronic pain has components of centralsensitization.
 6. The method of claim 1, wherein said chronic pain isneuropathic pain.
 7. The method of claim 1, wherein R¹ and each R⁶ is,independently, H, optionally substituted C₁₋₆ alkyl, optionallysubstituted C₁₋₄ alkaryl, optionally substituted C₁₋₄ alkheterocyclyl,or optionally substituted C₂₋₉ heterocyclyl.
 8. The method of claim 1,wherein R², R³, R⁴, or R⁵ has the formula:


9. The method of claim 8, wherein R^(2A) has the formula:

and each of X¹, X², X⁴, and X⁵ is independently selected from O, S, NR⁷,N, or CR⁸; X³ is selected from N or C; R⁷ is H or optionally substitutedC₁₋₆ alkyl; R⁸ is H, hal, optionally substituted C₁₋₆ alkyl, hydroxy,optionally substituted C₁₋₆ alkoxy, or optionally substituted C₁₋₆thioalkoxy, wherein at least one of X¹, X², X⁴, and X⁵ is not CR⁸. 10.The method of claim 9, wherein R^(2A) has the formula:

and each of X¹ and X² is independently selected from O, S, NH, N, or CH;and wherein at least one of X¹ and X² is not CH.
 11. The method of claim1, wherein said compound has a structure selected from

and wherein one of R⁴ and R⁵ has the following structure:

wherein X² is O or S.
 12. The method of claim 1, wherein Y¹ and Y² areeach H or together are ═O.
 13. The method of claim 1, wherein R¹ isoptionally substituted C₁₋₆ alkyl, optionally substituted C₂₋₉heterocyclyl, or optionally substituted C₁₋₄ alkheterocyclyl.
 14. Themethod of claim 13, wherein R¹ is optionally substituted aminoC₁₋₆alkylor optionally substituted C₁₋₄ alkheterocyclyl, wherein saidheterocyclyl is a 5- or 6-membered cyclic amine.
 15. The method of claim14, wherein said cyclic amine is substituted with a carboxyl, C₁₋₆alkoxycarbonyl, or carbamoyl group.
 16. The method of claim 13, whereinsaid heterocyclyl is optionally substituted pyrrolidinyl or optionallysubstituted piperidinyl.
 17. The method of claim 16, wherein R¹ is

wherein R⁹ is H, optionally substituted C₁₋₆ alkyl, or optionallysubstituted C₁₋₄ alkaryl.
 18. The method of claim 1, wherein R¹ is anoptionally substituted C₃-C₈ cycloalkyl.
 19. The method of claim 18,wherein said C₃-C₈ cycloalkyl is substituted by an amino.
 20. The methodof claim 1, wherein R¹ is —(CR^(1A)R^(1B))_(n)NR^(1C)R^(1D).
 21. Themethod of claim 20, wherein R^(1A) and R^(1B) are each H, and n is 2 or3.
 22. The method of claim 20, wherein R^(1C) is H, and R^(1D) is —CH₃,—CH₂CH₃, —(CH₂)₂OH, or —CH₂CO₂H; or R^(1C) and R^(1D) combine to formoptionally substituted pyrrolidinyl or optionally substitutedpiperidinyl.
 23. The method of claim 20, wherein R¹ is —CH₂CH₂N(CH₃)₂ or—CH₂CH₂NHCH₃.
 24. The method of claim 1, wherein one of R⁴ or R⁵ is H orF.
 25. A method of treating a condition selected from the groupconsisting of headache, neuropathic pain, chronic inflammatory pain,visceral pain, neuroinflammation, medication-induced hyperalgesia and/orallodynia, acute pain, chronic pain, and bone cancer pain in a mammal,wherein said method comprises administering an effective amount of thecompound selected from the group consisting of:

or a pharmaceutically acceptable salt thereof to said mammal.
 26. Themethod of claim 25, wherein said headache is migraine headache (with orwithout aura), chronic tension type headache (CTTH), migraine withallodynia, medication overuse headache, cluster headache, chronicheadache, or transformed migraine.